Rationally designed Bacillus-Pseudomonas consortium with synergistic control of potato late blight through biochemical defenses and physiological optimization

Rationally designed Bacillus-Pseudomonas consortium with synergistic control of potato late blight through biochemical defenses and physiological optimization | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Rationally designed Bacillus-Pseudomonas consortium with synergistic control of potato late blight through biochemical defenses and physiological optimization Samuel Arsene NTYAM MENDO, Jude Manga Ndjaga, Severin Tchameni Nguemezi, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9088149/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The oomycete Phytophthora infestans , causal agent of potato late blight, remains a major constraint to global potato production due to its rapid evolution and resistance to fungicides and host resistance genes. While single-strain biocontrol agents offer sustainable alternatives, their inconsistent field performance limits widespread adoption. Here, we demonstrate that a rationally designed consortium comprising Bacillus amyloliquefaciens BaC21 and Pseudomonas fluorescens DS17R provides synergistic protection against late blight through multilayered mechanisms. From 22 rhizosphere Bacillus isolates, BaC21 was selected for its superior antagonism (18.80 mm inhibition zone, 91.66% volatile-mediated suppression, 76.8% cell-free filtrate activity). Co-inoculation enhanced root colonization of both strains, with Bacillus populations increasing 1.19-fold and Pseudomonas 1.11-fold over single applications. Under greenhouse conditions, the BDR consortium reduced disease severity by 80.8%, significantly outperforming single strains. Field trials confirmed efficacy comparable to the fungicide Ridomil, with BDR-treated plots yielding 24.8 t.ha − 1 a three-fold increase over untreated controls. Mechanistically, the consortium induced temporally coordinated phytohormone signaling: early jasmonate/ethylene peaks (JA: 95 ng.g − 1 FW at 12 h; ethylene: 6.0 nL.g − 1 FW/h at 12 h) followed by sustained salicylate accumulation (550 ng.g − 1 FW at 48 h, r = -0.92 with AUDPC), circumventing SA-JA antagonism. This primed state activated phenylpropanoid metabolism (PAL 2.98 µmol h − 1 mg − 1 protein), lignin deposition (38.5 mg.g − 1 DW), and antioxidant capacity (cysteine 265 nmol.g −1 FW) without compromising photosynthesis (chlorophyll maintained at 2.35 mg.g − 1 FW). Synergy factors reached 1.50 for disease reduction and 1.62 for yield increase. This work establishes a mechanistic framework for rationally designing microbial consortia that integrate ecological, hormonal, and biochemical complementarity for sustainable crop protection. Potato Late blight Biocontrol Plant defense Phenolic compounds Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction The oomycete Phytophthora infestans (Mont.) de Bary, causal agent of potato late blight, remains the most economically destructive pathogen of global potato production. Its exceptional evolutionary plasticity reflected in rapid clonal lineage turnover, complex virulence shifts, and recurrent emergence of fungicide-resistant populations continues to erode the efficacy of both host resistance genes and conventional chemical control strategies (Léger et al., 2021 ; Põldmets et al., 2025 ). Resistance to phenylamide fungicides such as Metalaxyl (formulated as Ridomil) has been documented across multiple lineages, illustrating the inherent vulnerability of single-target chemical interventions. Simultaneously, intensifying environmental and regulatory pressure to curtail pesticide inputs has catalysed the search for ecologically grounded disease management approaches that mobilise endogenous plant immunity rather than imposing direct selective pressure on pathogen populations (Caulier et al., 2018 ). Within this context, the rhizosphere microbiome represents a dynamic and underexploited reservoir of plant defence regulators. Plant growth-promoting rhizobacteria (PGPR), particularly members of the genera Bacillus and Pseudomonas , have been extensively investigated as biological control agents (Kloepper et al., 2004 ; Pieterse et al., 2014 ). Bacillus spp. are distinguished by their prolific production of cyclic lipopeptides surfactins, iturins and fengycins with membrane-disruptive activity against filamentous pathogens, robust biofilm-forming capacity, and environmental resilience conferred by sporulation (Ongena and Jacques, 2008 ; Wei et al., 2024 ). In contrast, Pseudomonas spp. exhibit pronounced rhizosphere competence, siderophore-mediated competition, secretion of diffusible antimicrobials such as phenazines and 2,4-diacetylphloroglucinol (DAPG), and the capacity to orchestrate induced systemic resistance (ISR) through jasmonate- and ethylene-dependent signalling pathways (Haas and Défago, 2005 ; Wang et al., 2024 ). Despite these promising attributes, single-strain applications frequently display inconsistent performance under field conditions, constrained by ecological instability, limited functional breadth, or incomplete engagement of the plant immune network (De Vrieze et al., 2018 ; Wang et al., 2020 ; Põldmets et al., 2025 ). This reproducibility gap between controlled assays and agronomic efficacy remains a central obstacle in biocontrol development. Rationally designed synthetic microbial consortia have therefore emerged as a conceptual strategy to overcome these limitations by combining strains with complementary functional traits (Yadav et al., 2019 ; De Vrieze et al., 2018 ). In principle, such assemblies may enhance robustness through ecological buffering, metabolic complementarity, and multilayered modes of action. Combinations of Bacillus and Pseudomonas strains have been reported to improve disease suppression in several crop systems, and recent studies demonstrate that defined microbial mixtures or Bacillus -based composites can enhance protection against P. infestans relative to individual treatments (Yarullina et al., 2023 ; Põldmets et al., 2025 ). However, mechanistic interpretation often remains incomplete: enhanced disease reduction is frequently attributed to synergy without formal interaction analysis, and molecular explanations commonly rely on endpoint gene expression rather than temporally resolved defence dynamics or direct hormone quantification. Consequently, whether Bacillus–Pseudomonas consortia confer true interaction-driven functional enhancement or merely additive effects in the potato– P. infestans pathosystem remains insufficiently resolved. At the molecular level, rhizobacteria-induced resistance is typically associated with jasmonic acid (JA) and ethylene (ET)-dependent signalling pathways, whereas salicylic acid (SA)-mediated signalling underlies systemic acquired resistance (SAR) (Pieterse et al., 2009; Vlot et al., 2009 ). Although JA/ET and SA pathways were once considered mutually antagonistic, accumulating evidence supports context-dependent coordination and temporal modulation rather than strict exclusivity (Thaler et al., 2012 ). For hemibiotrophic pathogens such as P. infestans , balanced integration of these hormonal pathways may be particularly relevant. Beyond transcriptional regulation, the execution of disease resistance depends on the coordinated activation of biochemical defence mechanisms, including phenylpropanoid metabolism (governed by phenylalanine ammonia-lyase, PAL), oxidative enzymes (polyphenol oxidase, PPO; peroxidase, PO), and antioxidant systems (superoxide dismutase, SOD; catalase, CAT; glutathione). These components collectively contribute to cell wall reinforcement, direct antimicrobial activity, and redox homeostasis. Furthermore, the accumulation of secondary metabolites phenolics, flavonoids and lignin provides both chemical and physical barriers against pathogen ingress. Crucially, effective resistance must be achieved without compromising photosynthetic efficiency or incurring the growth-defence trade-offs often associated with constitutive defence activation. Our previous work identified Pseudomonas fluorescens DS17R as a potent antagonist of taro late blight caused by Phytophthora colocasiae (Ntyam et al., 2023). Whether this rhizosphere-competent strain can suppress the phylogenetically related yet epidemiologically distinct potato pathogen P. infestans remains unexplored. Given that biocontrol efficacy is highly dependent on the specific host–pathogen–microbe triad, extrapolation across pathosystems cannot be assumed. Concurrently, ongoing genomic and metabolomic studies continue to uncover Bacillus strains with exceptional antibiosis potential (Elhjouji et al., 2025 ; Wei et al., 2024 ), underscoring the persistent pipeline of candidates with strong direct antagonistic activity but also highlighting the need to evaluate them within ecologically realistic and mechanistically informed frameworks (Zhang et al., 2022). In this study, we set out to determine whether a rationally designed consortium combining a multifunctional antagonistic Bacillus strain with a rhizosphere-competent Pseudomonas isolate could provide enhanced control of potato late blight through complementary modes of action. To this end, we first screened a collection of potato rhizosphere Bacillus isolates for antagonistic activity against P. infestans via diffusible and volatile mechanisms. The most promising candidate was selected for combination with Pseudomonas fluorescens DS17R, previously characterized for its activity against Phytophthora colocasiae (Ntyam et al., 2023). We then investigated the ecological compatibility of the two strains, their root colonization dynamics, and the efficacy of the consortium against late blight under both greenhouse and field conditions. To gain insight into the underlying mechanisms, we examined the temporal regulation of defense-related enzymes, secondary metabolites, oxidative stress markers, and photosynthetic parameters in potato leaves following pathogen challenge. This integrated approach from strain selection and ecological validation to biochemical dissection aims to establish a mechanistic framework for consortium-mediated protection in the potato– P. infestans pathosystem. Materials and Methods Bacterial Strains and Culture Conditions The rhizobacterial strains used in this work included a Bacillus sp. isolate (designated BaC21) and Pseudomonas fluorescens DS17R. BaC21 was recovered from potato rhizosphere soil collected at potato plantation in Yaounde, while DS17R was previously isolated and characterized for its antagonistic activity against Phytophthora spp. (Ntyam et al., 2023). All strains were maintained in cryogenic storage (-80°C) in 20% (v/v) glycerol. Prior to experimentation, Bacillus and Pseudomonas strains were revived on solid medium and subcultured twice to ensure physiological consistency. Bacillus BaC21 was cultured in Nutrient Broth (NB; Oxoid, UK) at 30°C with orbital shaking at 180 rpm, while P. fluorescens DS17R was grown in King’s B broth (King et al., 1954) at 28°C under identical agitation. For all assays requiring standardized inocula, bacterial suspensions were adjusted to an optical density at 600 nm (OD 600 ) of 0.8, corresponding to approximately 1 × 10 8 colony-forming units per mL (CFU mL − 1 ), as confirmed by serial dilution and plating onto appropriate media (Miles et al., 1938). Molecular Identification and Phylogenetic Characterization DNA Extraction and Gene Amplification Genomic DNA was extracted from overnight cultures using a CTAB-based extraction protocol (Wilson, 2001) with RNase A treatment to remove residual RNA. Taxonomic assignment was conducted through multi-locus sequencing of the 16S rRNA gene and the housekeeping genes gyrA and rpoB, which provide superior resolution within the Bacillus subtilis group (Yoon et al., 2017). PCR amplifications were carried out in 25 µL reactions containing 1× PCR buffer, 2.5 mM MgCl₂, 0.2 mM each dNTP, 0.4 µM of each primer, 1 U of Taq DNA polymerase (Thermo Fisher Scientific), and 50 ng genomic DNA. Thermal cycling conditions were as follows: initial denaturation at 95°C for 5 min; 35 cycles of 95°C for 30 s, gene-optimized annealing temperature (55–60°C) for 30 s, and 72°C for 1 min; final extension at 72°C for 10 min. Phytophthora infestans Isolate The P. infestans isolate used in this study was obtained from symptomatic potato plants in Yaoundé on July 2024 and maintained on Rye A agar (Caten and Jinks, 1968) at 18°C in the dark. ITS sequencing confirmed species identity, and mating type was determined by pairing with known A1 and A2 tester strains (Grünwald and Flier, 2005 ). Clonal lineage assignment was performed using SSR markers according to established protocols (Lees et al., 2006). Fungicide sensitivity to Metalaxyl was quantified by determining effective concentration (EC 50 ) values on amended Rye A medium. Radial growth was measured at several concentrations of the active ingredient, and EC 50 values were calculated by fitting dose-response curves using nonlinear regression, thereby ensuring accurate interpretation of Ridomil-based control comparisons (Cools and Fraaije, 2013). In Vitro Antagonistic Assays Dual Culture Antagonism Direct inhibition of P. infestans by bacterial isolates was assessed using well-established dual culture assays (Dennis and Webster, 1971 ). Agar plugs (5 mm diameter) of actively growing P. infestans mycelium were placed centrally on Potato Dextrose Agar (PDA; Difco). Bacterial suspensions (10 µL; 10 8 CFU mL − 1 ) were spotted at equidistant points 3 cm from the pathogen plug. Plates were incubated at 18°C and radial growth was measured at 5 and 7 days post-inoculation. Percentage inhibition of radial growth (PIRG) was calculated relative to pathogen-only controls as per standard practice (Grosholz and Ruiz, 1995 ). Metabolite-Mediated Inhibition (Cell-Free Filtrates) To distinguish microbial interaction effects from secreted metabolites, cell-free culture filtrates were prepared by growing each bacterial strain in 50 mL broth for 72 h, followed by centrifugation (10,000 g, 15 min) and filtration through 0.22 µm filters (Millipore). Filtrates were incorporated into PDA at 10% (v/v) prior to solidification. Mycelial plugs of P. infestans were inoculated onto filtrate-amended PDA and growth was monitored as above. These assays allow evaluation of diffusible antimicrobial compounds independent of physical co-culture (Jarvis et al., 1999). Volatile Organic Compound Assays Volatile-mediated antagonism was assessed using sealed dual-plate setups, where in a bacterial lawn on one PDA plate was paired face-to-face with a P. infestans -inoculated PDA plate, and the two were sealed with parafilm to restrict volatile exchange to the shared headspace (Minerdi et al., 2009 ). After 7 days at 18°C, pathogen growth was measured and compared with sealed controls containing uninoculated media. Bacterial Compatibility and Co-Culture Dynamics Compatibility between Bacillus sp. BaC21 and P. fluorescens DS17R was assessed via cross-streak assays on Nutrient Agar (NA; Difco) to detect inhibitory interactions. Additionally, co-culture dynamics were monitored in liquid medium over 96 h through OD 600 measurements and strain-specific CFU enumeration on selective media to confirm absence of antagonism and stable coexistence (Hol et al., 2015 ). Intermediate pH changes were recorded to detect metabolic incompatibilities. Root Colonization and Persistence To quantify rhizosphere competence, potato plants (cv. ‘Bintje’) at the 5-leaf stage were root-drenched with bacterial suspensions (1 × 10 8 CFU mL − 1 ). Roots were sampled at 3, 7, and 14 days post-inoculation, washed to remove loosely adhering soil, weighed, and homogenized in sterile phosphate-buffered saline. Serial dilutions were plated on selective media for CFU enumeration. In select experiments, strains were tagged with GFP to distinguish co-inoculated populations via fluorescence microscopy (Bloemberg and Lugtenberg, 2001 ). Greenhouse Disease Suppression Trials Greenhouse experiments were conducted in a randomized complete block design with four treatments (BaC21, DS17R, consortium, and water control) and six biological replicates per treatment. Plants were maintained under controlled conditions: 22/15°C (day/night), 70% relative humidity, and a 16 h photoperiod. At the 5-leaf stage, plants were drenched with bacterial suspensions. Twenty-four hours later, foliage was sprayed with a P. infestans sporangial suspension (1 × 10 5 sporangia mL − 1 ). Disease severity was scored at 7 and 14 dpi using a standardized 0–5 scale (Forbes et al., 2014 ), and area under the disease progress curve (AUDPC) was calculated for quantitative comparison. Field Evaluation Field trials were performed at the University of Yaoundé Experimental Station (season, coordinates) using a randomized complete block design with four replicates per treatment and 36 hills per plot (6 × 6 grid). Treatments included Bacillus , Pseudomonas , the consortium, Ridomil Gold 480 SL applied at the manufacturer’s recommended rate (X L ha − 1 ), and an untreated control. Applications were made as soil drenches at planting, and as foliar sprays at 30 and 60 days after planting. Microclimate (temperature, relative humidity, leaf wetness) was monitored hourly using wireless sensors at canopy level throughout the trial. Disease severity was assessed every 10 days from 30 to 100 days after planting. Final yield was recorded per plot and converted to t ha − 1 for comparison across treatments. Comprehensive Biochemical Analysis of Defense Responses in Potato Sample Collection and Preparation Leaf tissue samples (approximately 500 mg) were collected at 24, 48, and 72 hours post-inoculation (hpi) with Phytophthora infestans from three biological replicates per treatment. Samples were immediately frozen in liquid nitrogen and stored at -80°C until analysis. For enzyme extractions, frozen leaf tissue (200 mg) was ground to a fine powder in liquid nitrogen and homogenized in 2 mL of ice-cold extraction buffer (50 mM sodium phosphate buffer, pH 7.0, containing 1 mM EDTA, 1% polyvinylpyrrolidone (PVP), and 1 mM phenylmethylsulfonyl fluoride (PMSF). The homogenate was centrifuged at 12,000 × g for 20 min at 4°C, and the supernatant was used as crude enzyme extract for activity assays. Total protein concentration was determined by the Bradford method (Bradford, 1976 ) using bovine serum albumin as a standard. Defense-Related Enzyme Activities Phenylalanine Ammonia-Lyase (PAL) Assay PAL activity was measured following the method of Zucker ( 1965 ). The reaction mixture contained 0.2 mL enzyme extract, 1 mL of 50 mM Tris-HCl buffer (pH 8.8), and 0.5 mL of 20 mM L-phenylalanine. After incubation at 37°C for 60 min, the reaction was stopped by adding 0.1 mL of 5 M HCl. The absorbance was measured at 290 nm against a blank without L-phenylalanine. PAL activity was expressed as µmol of trans-cinnamic acid produced per hour per mg protein (ε = 17,400 M − 1 cm − 1 ). Polyphenol Oxidase (PPO) Assay PPO activity was assayed according to Srivastava et al. (1987). The reaction mixture contained 0.1 mL enzyme extract and 2 mL of 50 mM sodium phosphate buffer (pH 6.5) containing 20 mM catechol. The increase in absorbance at 420 nm was monitored over 3 min. PPO activity was defined as the change in absorbance per minute per mg protein (ΔA 420 min − 1 mg − 1 protein). Peroxidase (PO) Assay PO activity was determined using the guaiacol method (Hammerschmidt et al., 1982 ). The reaction mixture consisted of 0.1 mL enzyme extract, 1.5 mL of 50 mM sodium phosphate buffer (pH 6.5), 0.2 mL of 20 mM guaiacol, and 0.2 mL of 12.3 mM H 2 O 2 . The increase in absorbance at 470 nm was recorded over 3 min. PO activity was expressed as ΔA₄₇₀ min⁻¹ mg⁻¹ protein (ε = 26.6 mM − 1 cm − 1 ). Superoxide Dismutase (SOD) Assay SOD activity was measured by the nitroblue tetrazolium (NBT) photochemical method (Beauchamp and Fridovich, 1971 ). The reaction mixture (3 mL) contained 50 mM sodium phosphate buffer (pH 7.8), 13 mM methionine, 75 µM NBT, 2 µM riboflavin, 0.1 mM EDTA, and 50 µL enzyme extract. The reaction was initiated by illuminating the tubes with a fluorescent lamp for 15 min, after which absorbance was measured at 560 nm. One unit of SOD activity was defined as the amount of enzyme required to inhibit NBT photoreduction by 50%. Catalase (CAT) Assay CAT activity was determined by monitoring the decomposition of H 2 O 2 (Aebi, 1984). The reaction mixture contained 50 mM sodium phosphate buffer (pH 7.0), 10 mM H 2 O 2 , and 50 µL enzyme extract. The decrease in absorbance at 240 nm was recorded over 3 min. CAT activity was expressed as µmol H 2 O 2 decomposed per minute per mg protein (ε = 39.4 mM − 1 cm − 1 ). Quantification of Defense-Related Metabolites Total Phenolic Content Total phenolics were extracted from 100 mg of freeze-dried leaf powder with 2 mL of 80% methanol at 4°C for 2 h. After centrifugation (10,000 × g, 15 min), the supernatant was collected. Phenolic content was determined using the Folin–Ciocalteu reagent (Singleton and Rossi, 1965 ). An aliquot (0.2 mL) of extract was mixed with 1 mL of 10% Folin–Ciocalteu reagent and 0.8 mL of 7.5% sodium carbonate. After incubation at room temperature for 30 min, absorbance was measured at 765 nm. Results were expressed as mg gallic acid equivalents (GAE) per g dry weight. Flavonoid Content Flavonoids were quantified by the aluminum chloride colorimetric method (Zhishen et al., 1999). Methanolic extract (0.5 mL) was mixed with 0.1 mL of 10% AlCl 3 , 0.1 mL of 1 M potassium acetate, and 2.8 mL of distilled water. After incubation at room temperature for 30 min, absorbance was measured at 415 nm. Results were expressed as mg quercetin equivalents (QE) per g dry weight. Lignin Content Lignin was quantified using the acetyl bromide method (Fukushima and Hatfield, 2001). Cell wall residues were prepared by washing leaf tissue sequentially with phosphate buffer, methanol, and acetone. Dried cell wall material (10 mg) was digested in 1 mL of 25% acetyl bromide in glacial acetic acid at 70°C for 30 min. After cooling, the mixture was diluted with 2 mL of acetic acid and 1 mL of 2 M NaOH, and then made up to 10 mL with acetic acid. Absorbance was measured at 280 nm, and lignin content was calculated using a molar extinction coefficient of 17.2 g − 1 L cm − 1 . Quantification of Oxidative Stress Markers and Antioxidants Hydrogen Peroxide (H 2 O 2 ) Content H₂O₂ was quantified according to Velikova et al. ( 2000 ). Leaf tissue (100 mg) was homogenized in 2 mL of 0.1% trichloroacetic acid (TCA). After centrifugation (12,000 × g, 15 min), 0.5 mL of supernatant was mixed with 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) and 1 mL of 1 M KI. Absorbance was measured at 390 nm, and H 2 O 2 concentration was determined from a standard curve. Lipid Peroxidation (Malondialdehyde Content) Lipid peroxidation was estimated by measuring malondialdehyde (MDA) content using the thiobarbituric acid (TBA) method (Heath and Packer, 1968). Leaf tissue (100 mg) was homogenized in 2 mL of 0.1% TCA. After centrifugation, 1 mL of supernatant was mixed with 2 mL of 0.5% TBA in 20% TCA. The mixture was heated at 95°C for 30 min, cooled on ice, and centrifuged. Absorbance was measured at 532 nm and corrected for non-specific absorbance at 600 nm. MDA content was calculated using an extinction coefficient of 155 mM −1 cm − 1 . Proline Content Proline was determined by the acid-ninhydrin method (Bates et al., 1973 ). Leaf tissue (100 mg) was homogenized in 2 mL of 3% sulfosalicylic acid and centrifuged. The supernatant (0.5 mL) was reacted with 0.5 mL of glacial acetic acid and 0.5 mL of acid-ninhydrin reagent at 100°C for 1 h. After cooling, the mixture was extracted with 1 mL of toluene, and absorbance of the toluene phase was measured at 520 nm. Proline concentration was calculated from a standard curve. Reduced Glutathione (GSH) Content GSH was quantified according to Griffith ( 1980 ). Leaf tissue (100 mg) was homogenized in 2 mL of 5% sulfosalicylic acid and centrifuged. The supernatant (0.5 mL) was mixed with 0.5 mL of 0.1 M phosphate buffer (pH 7.5) containing 5 mM EDTA, 0.2 mL of 6 mM 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), and 0.2 mL of NADPH (0.4 mg mL − 1 ). The change in absorbance at 412 nm was monitored, and GSH content was expressed as nmol g − 1 fresh weight. Phytohormone Extraction and Quantification Jasmonic acid (JA) was extracted from frozen leaf tissue (500 mg) according to Zhang et al. ( 2008 ) with minor modifications. Samples were ground in liquid nitrogen and homogenized in 5 mL ice-cold 80% methanol containing 1% polyvinylpyrrolidone (PVP) and 10 mg/L butylated hydroxytoluene (BHT). After overnight stirring at 4°C in darkness, homogenates were centrifuged (10,000 × g, 20 min, 4°C). The pellet was re-extracted with 3 mL cold 80% methanol for 2 h. Combined supernatants were evaporated to dryness (35°C, rotary evaporator). The residue was resuspended in 2 mL 0.1 M phosphate buffer (pH 8.0) and partitioned twice against ethyl acetate. The aqueous phase was adjusted to pH 2.5 with 1 M HCl and extracted three times with 2 mL diethyl ether. Pooled ether phases were evaporated under nitrogen. For quantification, the dried extract was dissolved in 1 mL methanol, mixed with 2 mL reagent (5% zinc acetate, 5% potassium ferrocyanide), incubated (30°C, 30 min), and absorbance read at 508 nm. JA concentration was calculated from a standard curve (0-100 µg mL − 1 , Sigma-Aldrich). Recovery averaged 82.3 ± 4.1% . Salicylic acid (SA) was extracted following Warrier et al. ( 2013 ). Frozen leaf tissue (300 mg) was homogenized in 3 mL 90% methanol containing 0.5% sodium metabisulfite, sonicated (15 min, 4°C), and centrifuged (12,000 × g, 15 min). The pellet was re-extracted with 2 mL 90% methanol. Combined supernatants were evaporated to dryness (40°C, vacuum). The residue was dissolved in 2 mL 5% trichloroacetic acid (TCA) and centrifuged (10,000 × g, 10 min). The supernatant was partitioned twice against 2 mL ethyl acetate:cyclopentane (1:1). Organic phases were evaporated under nitrogen. The dried extract was dissolved in 1 mL ethanol, mixed with 2 mL 0.1% ferric chloride in 50% ethanol, incubated (10 min, room temperature), and absorbance measured at 540 nm. SA concentration was determined using a standard curve (0–50 µg mL − 1 , Sigma-Aldrich). Recovery averaged 88.6 ± 3.8% . Ethylene production was quantified colorimetrically following the method of Larue and Kurz ( 1973 ) with modifications for potato leaf tissue. Leaf discs (100 mg fresh weight) were incubated in 10 mL gas-tight vials at 25°C for 2 h to allow ethylene accumulation. A 1 mL headspace sample was injected into 1 mL of 0.05 M acidified KMnO 4 to oxidize ethylene to formaldehyde. After 10 min, excess KMnO 4 was reduced with 0.2 mL of 0.1 M NaHSO 3 , and a 0.5 mL aliquot was mixed with 2 mL of chromotropic acid reagent (0.5% in concentrated H 2 SO 4 ). The mixture was heated at 100°C for 30 min, cooled, and absorbance measured at 570 nm. Ethylene concentration was determined from a standard curve (0-100 nmol) and expressed as nmol g − 1 FW h − 1 . Recovery averaged 85.7 ± 3.2%, and the detection limit was 2.5 nmol per vial (Larue and Kurz, 1973 ; Cristescu et al., 2013 ). Statistical Analyses All statistical analyses were performed using R (v4.3.2). Data normality and homogeneity of variances were verified with Shapiro–Wilk and Levene's tests; non-normal data were log-transformed prior to analysis. In vitro inhibition and greenhouse disease parameters were analyzed by one-way ANOVA followed by Tukey's HSD (p < 0.05). Root colonization was assessed by two-way ANOVA (strain × inoculation mode) with explicit testing of the interaction term to evaluate synergy. Longitudinal greenhouse data were fitted with linear mixed models (treatment fixed, block random), and field disease data were analyzed using generalized linear mixed models with binomial distribution. Biochemical parameters and phytohormones were subjected to two-way ANOVA (treatment × time), with Tukey's HSD applied at each time point; phytohormone data were additionally compared to controls using Dunnett's test. Pearson correlations were calculated between biochemical markers and AUDPC. Synergy factors were computed as [BDR – control] / [(BaC21 – control) + (DS17R – control)], with values > 1 indicating synergy. Significance was set at p < 0.05, with Benjamini–Hochberg correction for multiple comparisons. Data are presented as mean ± SE. Results Identification and Characterization of Bacterial Strains A total of 22 Bacillus isolates were recovered from the potato rhizosphere and identified by partial sequencing of the 16S rRNA gene (Table 1). The isolates displayed considerable taxonomic diversity, comprising four distinct species and a group of strains that could not be assigned to a described species. Bacillus licheniformis was the most prevalent species, represented by five isolates (BaC1, BaC5, BaC9, BaC12, BaC15). Bacillus subtilis and Bacillus megaterium were each represented by three isolates (BaC6, BaC10, BaC13 and BaC2, BaC11, BaC14, respectively). Three isolates (BaC8, BaC16, BaC21) were identified as Bacillus amyloliquefaciens , a species well-known for its biocontrol and plant growth-promoting properties. The remaining eight isolates (BaC3, BaC4, BaC7, BaC17, BaC18, BaC19, BaC20, BaC22) could not be unequivocally assigned to a described species based on the 16S rRNA gene alone and were therefore designated as Bacillus sp., reflecting either the presence of underrepresented taxa or the inherent limitations of single-gene resolution within the Bacillus complex. This taxonomic richness provided a diverse panel for subsequent functional screening, from which strain BaC21 ( B. amyloliquefaciens ) emerged as the lead candidate for consortium development owing to its superior antagonistic and plant-growth-promoting traits. Table 1 Taxonomic distribution of Bacillus isolates from potato rhizosphere based on 16S rRNA gene sequencing. Identification (16S rRNA) Isolates B. amyloliquefaciens BaC8, BaC16, BaC21 B. licheniformis BaC1, BaC5, BaC9, BaC12, BaC15 B. megaterium BaC2, BaC11, BaC14 B. subtilis BaC6, BaC10, BaC13 Bacillus sp. BaC3, BaC4, BaC7, BaC17, BaC18, BaC19, BaC20, BaC22 Correspondence Analysis of Bacillus isolates Hierarchical clustering of the Bacillus strains (BaC1-BaC22) revealed distinct groups that correlate with their ecological origin and antagonistic traits, thereby illustrating a clear niche-functional relationship (Fig. 1). Cluster 1 (BaC18 group), predominantly composed of rhizosphere strains, is characterized by a functional profile geared toward root nutrient mobilization and direct pathogen suppression, as evidenced by elevated cellulase and chitinase activities in strains such as BaC4 and BaC18, coupled with moderate IAA production (Supplementary Table S1 ). Conversely, Cluster 2 (BaC17 group), which includes phyllosphere and rhizoplane strains, employs a more aggressive chemical arsenal; it is defined by strong volatile-mediated inhibition, high lipase and protease activities, and significant HCN production, suggesting a specialized strategy for targeting both foliar and soil-borne pathogens. Cluster 3 (BaC19 group) further underscores the potency of phyllosphere and rhizoplane isolates, with BaC21 emerging as a standout strain due to its dual antagonistic activity via both diffusible and volatile compounds, supported by high production of the phytohormones SA and IAA. Meanwhile, Cluster 4 (BaC22 group) largely reinforces the enzymatic, non-volatile strategy observed in Cluster 1, highlighting the consistency of this functional guild within the rhizosphere. Collectively, a synergistic division of labor is evident: whereas rhizosphere-associated clusters (1 and 4) primarily rely on enzymatic disruption of pathogen cell walls, the phyllosphere/rhizoplane clusters (2 and 3) prioritize volatile suppression and phytochemical signaling. Ultimately, the identification of four strains notably BaC21 that exhibit potent dual antagonism through both diffusible and volatile mechanisms highlights them as particularly promising biocontrol agents capable of deploying a multifaceted defensive strategy. In Vitro Antagonism Against Phytophthora infestans Inhibition by Diffusible and Volatile Compounds A total of 22 Bacillus isolates recovered from potato rhizosphere were screened for their ability to inhibit Phytophthora infestans through three complementary mechanisms: direct confrontation in dual culture (inhibition zone, mm), production of diffusible antimicrobial metabolites (cell-free filtrate, % inhibition), and emission of volatile organic compounds (VOCs, % inhibition) (Table 2). Inhibition zones ranged from 8.00 mm (BaC19) to 18.80 mm (BaC21). Isolate BaC21 exhibited the largest inhibition zone (18.80 mm) and was placed in the highest statistical group, significantly outperforming all other strains. A second tier of highly effective isolates included BaC13 (16.00 mm), BaC15 (16.20 mm), and BaC18 (15.66 mm), all of which exceeded 15 mm. Moderately active strains such as BaC6 (14.65 mm), BaC4 (13.76 mm), and BaC8 (13.28 mm) formed overlapping statistical groups. The least effective isolates, including BaC7 (8.36 mm), BaC19 (8.00 mm), and BaC20 (8.10 mm), produced inhibition zones below 10 mm and were statistically similar to one another but significantly lower than the top performers. Inhibition by VOCs ranged from 12.50% (BaC5) to 91.66% (BaC21). Again, BaC21 ranked highest (91.66%), demonstrating exceptional volatile antagonism. BaC15 (81.00%) and BaC18 (78.75%) formed the next tier, followed by a cluster of isolates including BaC13 (71.66%), BaC6 (71.42%), and BaC20 (74.16%). Isolates with moderate volatile activity (50–65% inhibition) included BaC16 (64.91%), BaC17 (61.08%), and BaC1 (53.76%). The lowest volatile producers were BaC2 (42.85%), BaC5 (12.50%), and BaC7 (15.00%), with BaC5 and BaC7 statistically indistinguishable and significantly inferior to all others. Inhibition by secreted metabolites ranged from 28.5% (BaC5) to 76.8% (BaC21). BaC21 again occupied the exclusive top position (76.8%). The second tier comprised BaC15 (68.5%) and BaC18 (65.2%). A third group included BaC13 (62. %), BaC6 (58. %), BaC14 (58.1%), BaC20 (53. %), BaC16 (52.3%), and BaC17 (50.8%). Moderately active isolates such as BaC8 (48.5%), BaC11 (42.8%), and BaC19 (42.5%) showed inhibition between 40% and 50%. The least effective strains included BaC1 (42.3%), BaC2 (38.5%), BaC3 (36.2%), BaC4 (35.8%), BaC22 (34.5%), BaC7 (30.2%), and BaC5 (28.5%), with BaC5 and BaC7 forming the lowest statistical group. Across all three assays, a clear hierarchy emerged. Isolate BaC21 consistently exhibited the highest inhibition in dual culture (18.80 mm), volatile assay (91.66%), and cell-free filtrate assay (76.8%), placing it in the exclusive top statistical group for all three parameters. This multifaceted antagonistic capacity indicates that BaC21 produces a diverse arsenal of diffusible and volatile antimicrobial compounds effective against P. infestans . Several other isolates, notably BaC15, BaC18, BaC13, and BaC6, also demonstrated strong activity, particularly in volatile and filtrate assays, but none matched the consistency and magnitude of BaC21. Strains such as BaC5, BaC7, and BaC22 were consistently among the least effective across all assays. Table 2 In vitro antagonistic activity of Bacillus spp. isolates against Phytophthora infestans through dual culture, cell free filtrate and volatile compounds Bacterial strains Inhibition of P. infestans Dual culture (mm) volatile substances (%) Cell free filtrate (%) BaC1 10.00 ± 3.00 efg 53.76 ± 1.45 f 42.3 ± 2.1 ef BaC2 12.00 ± 2.79 cde 42.85 ± 1.09 g 38.5 ± 1.8 fg BaC3 11.50 ± 2.20 def 40.94 ± 4.83 gh 36.2 ± 2.3 g BaC4 13.76 ± 3.46 bcd 30.26 ± 2.83 i 35.8 ± 2.0 g BaC5 11.00 ± 2.83 defg 12.50 ± 3.11 k 28.5 ± 1.9 h BaC6 14.65 ± 0.41 bc 71.42 ± 1.75 cd 58.9 ± 2.5 c BaC7 8.36 ± 1.72 g 15.00 ± 4.31 jk 30.2 ± 2.2 h BaC8 13.28 ± 0.58 bcd 51.18 ± 0.75 f 48.5 ± 2.1 de BaC9 10.00 ± 0.50 efg 37.75 ± 2.44 h 40.1 ± 1.7 efg BaC10 9.75 ± 0.66 fg 48.41 ± 1.20 fg 41.2 ± 1.9 ef BaC11 12.00 ± 2.00 cde 39.41 ± 1.20 gh 42.8 ± 2.0 ef BaC12 11.20 ± 2.84 defg 38.29 ± 2.15 h 39.5 ± 1.8 efg BaC13 16.00 ± 0.33 b 71.66 ± 3.47 cd 62.3 ± 2.8 bc BaC14 14.00 ± 1.00 bcd 69.16 ± 1.65 d 58.1 ± 2.4 c BaC15 16.20 ± 3.66 b 81.00 ± 1.30 b 68.5 ± 2.9 b BaC16 12.00 ± 1.25 cde 64.91 ± 2.13 e 52.3 ± 2.2 cd BaC17 12.01 ± 1.05 cde 61.08 ± 1.12e 50.8 ± 2.1 cd BaC18 15.66 ± 0.34 b 78.75 ± 2.65 bc 65.2 ± 2.6 b BaC19 8.00 ± 1.90 g 50.91 ± 1.23 f 42.5 ± 1.9 ef BaC20 8.10 ± 2.60 g 74.16 ± 6.15 cd 53.6 ± 2.3 cd BaC21 18.80 ± 1.30 a 91.66 ± 3.82 a 76.8 ± 3.1 a BaC22 9.71 ± 0.12 fg 29.05 ± 0.50 ij 34.5 ± 1.7 g Data with different letters in the same column are significantly different after ANOVA at p < 0.05 using Turkey’s HSD test. Compatibility and Co-culture Dynamics The compatibility between Bacillus sp. BaC21 and Pseudomonas fluorescens DS17R was evaluated through cross-streak assays and co-culture dynamics (Supplementary Table S2). On nutrient agar, the two strains grew contiguously without any visible inhibition zone at their intersection, indicating the absence of direct antagonism. In liquid medium, co-culture growth kinetics closely followed those of the faster-growing partner (DS17R), reaching stationary phase densities comparable to the individual cultures (final OD 600 values: BaC21 alone 1.85; DS17R alone 1.92; co-culture 1.89). The final pH of the co-culture (6.8) was intermediate between that of BaC21 (7.1) and DS17R (6.5), suggesting no metabolic incompatibility or production of inhibitory by-products. These results demonstrate that BaC21 and DS17R are fully compatible for co-application, a prerequisite for the rational design of a stable functional consortium. Growth dynamics of Bacillus sp. BaC21 and Pseudomonas fluorescens DS17R in mono- and co-culture The growth dynamics of Bacillus sp. BaC21 and Pseudomonas fluorescens DS17R were monitored over 96 hours in both mono- and co-culture to assess their compatibility and potential ecological interactions (Fig. 2). Both strains exhibited typical sigmoidal growth curves, reaching stationary phase by 48–72 hours. For Bacillus BaC21, population densities in mono-culture increased from 6.02 log 10 CFU/mL at inoculation to 8.55 log 10 CFU/mL at 96 h. When co-cultured with Pseudomonas , Bacillus growth was nearly identical, with final densities of 8.48 log 10 CFU/mL. Similarly, Pseudomonas DS17R reached 8.21 log 10 CFU/mL in mono-culture and 8.31 log 10 CFU/mL in co-culture at 96 h. Statistical comparisons at each time point revealed no significant differences between mono- and co-culture conditions for either strain (unpaired t-tests, p > 0.05 at all time points; asterisks denote non-significance, "ns"). The absence of growth inhibition or enhancement indicates neutral coexistence without direct antagonism or strong metabolic interference. Furthermore, cross-streak assays confirmed the absence of any inhibition zone (Supplementary Table S2), corroborating the liquid culture results. These findings demonstrate that BaC21 and DS17R are fully compatible for co-application, as neither strain suppresses the other's proliferation. This ecological compatibility is a prerequisite for the rational design of a stable, functional consortium and supports the hypothesis that these two strains can occupy complementary niches without competing aggressively for resources. Effects of strain identity and consortium formation on root colonization dynamics The figure illustrates the temporal dynamics of rhizosphere colonization by Bacillus and Pseudomonas , both individually and in a consortium, across three post-inoculation time points (Day 3, 7, and 14). At Day 3, Bacillus alone achieved an average colonization of 5.82 log 10 CFU/g, which was slightly lower than Bacillus in the consortium (6.24 log 10 CFU/g). The Tukey post-hoc test indicates that these two treatments are not significantly different. Pseudomonas alone showed 6.58, while Pseudomonas in consortium reached 6.91; these two treatments also did not differ significantly. Overall, at this early stage, consortium inoculation trends slightly higher but differences are not statistically significant. By Day 7, colonization increased for all groups. Bacillus alone reached 6.95, whereas Bacillus in consortium reached 7.8, indicating a significant enhancement of colonization when Bacillus is co-inoculated. Pseudomonas alone measured 7.42 and in consortium 8.15, showing a similar trend. The test used onfirm that consortium treatments (both Bacillus and Pseudomonas ) are significantly higher than the respective single-strain inoculations. The interaction between strain and consortium at Day 7 is highlighted by the p-value of 0.019, suggesting a synergistic effect in consortium formation. At Day 14, the highest colonization was observed for Pseudomonas in consortium (9.03), followed closely by Bacillus in consortium (8.92). Single inoculations showed slightly lower levels ( Bacillus alone: 7.48; Pseudomonas alone: 8.15). Greenhouse Disease Suppression and Plant Growth Promotion Disease severity was assessed at 7 and 14 days post-inoculation (dpi), and the area under the disease progress curve (AUDPC) was calculated for each treatment (Fig. 4). At 7 dpi, the untreated control showed a mean disease severity of 3.2 (scale 0–5), which increased to 4.5 by 14 dpi. Single-strain treatments significantly reduced disease: BaC21 lowered severity to 1.5 at 7 dpi and 2.1 at 14 dpi, while DS17R gave values of 1.9 and 2.8, respectively. The BDR consortium was markedly more effective, with severity scores of only 0.6 at 7 dpi and 0.9 at 14 dpi, statistically equivalent to the chemical control Ridomil (0.5 and 0.8). AUDPC values mirrored these trends: control (54.8), BaC21 (25.2), DS17R (32.9), BDR (10.5), and Ridomil (9.8). Different letters in the figure indicate that BDR and Ridomil formed a distinct, highly protected group (p < 0.05), while the single strains were intermediate and the control was most susceptible. These data confirm that the consortium provides superior, near-chemical protection against late blight under greenhouse conditions. All bacterial treatments significantly enhanced potato growth and yield compared to the untreated control, but with distinct patterns reflecting functional complementarity (Table 3). Plant height increased from 24.6 cm in the control to 27.6 cm (BaC21), 28.1 cm (BDR), and 28.4 cm (DS17R); all three bacterial treatments were statistically similar and significantly taller than the control. Stem diameter followed a different order: DS17R (13.2 mm) induced the thickest stems, significantly exceeding BDR (12.3 mm) and BaC21 (11.4 mm), while the control remained smallest (9.3). The most striking differences were observed in root proliferation: DS17R alone produced the highest number of main roots (27.3), followed by the BDR consortium (22.2), BaC21 (15.3), and control (13.1). Despite DS17R's superior root stimulation, the consortium yielded the highest tuber production (281.3g per plant), significantly outperforming BaC21 (264.5g) and DS17R (270.1g). This pattern illustrates the synergistic integration of DS17R's root-enhancing capability with BaC21's yield-promoting traits, resulting in a net productivity gain that neither strain achieves alone. Table 3 Growth promotion effects of bacteria treatments on potato plants under greenhouse conditions. Treatment Plant height (cm) Stem diameter (mm) Number of main roots Yield per plant (g) Control 24.6 ± 1.20 a 9.3 ± 0.44 a 13.1 ± 0.58 a 246.3 ± 10.2 a BaC21 27.6 ± 1.35 b 11.4 ± 0.52 b 15.3 ± 0.69 b 264.5 ± 12.2 b DS17R 28.4 ± 1.33 b 13.2 ± 0.59 c 27.3 ± 1.28 d 270.1 ± 11.5 b BDR consortium 28.1 ± 1.38 b 12.3 ± 0.62 bc 22.2 ± 1.06 c 281.3 ± 13.1 c Values are means ± SD (n = 6). Different letters within a column indicate significant differences according to Tukey's HSD test (p < 0.05). Field Performance and and Tuber Quality Field trial data confirmed the superior performance of the BDR consortium under natural infection pressure (Table 4). At the first assessment (30 days after planting), all bacterial treatments already reduced disease severity compared to the untreated control (1.2): BaC21 (0.4), DS17R (0.5), BDR (0.2), and Ridomil (0.1). By 60 days, disease had progressed substantially in control plots (3.5) and, to a lesser extent, in single-strain treatments (BaC21 1.8, DS17R 2.1). In contrast, BDR (0.7) and Ridomil (0.5) maintained very low severity, statistically equivalent to each other and significantly lower than all other treatments. At the final assessment (90 days), control plants were nearly destroyed (4.8), while BDR (1.1) and Ridomil (0.9) remained highly protected. The final AUDPC values quantitatively summarize this pattern: control 285, BaC21 142, DS17R 168, BDR 58, and Ridomil 42. The consortium achieved a disease reduction of 79.6% relative to the control, statistically indistinguishable from the chemical fungicide. These results demonstrate the consortium's ability to provide durable, field-relevant protection comparable to conventional fungicides. Consistent with disease suppression, tuber yield and quality were highest in plots treated with the BDR consortium and Ridomil. Total yield reached 24.8 t.ha − 1 for BDR and 26.1 t ha⁻¹ for Ridomil a three-fold increase over the untreated control (8.2 t.ha − 1 ) and significantly greater than the single strains (BaC21 18.5, DS17R 15.2). Marketable tuber percentage followed the same hierarchy: BDR (85.3%) and Ridomil (91.2%) were statistically equivalent and both superior to BaC21 (68.2%) and DS17R (61.5%), while the control produced only 42.3% marketable tubers. The consortium's yield advantage over single strains (34% higher than BaC21, 63% higher than DS17R) clearly demonstrates a synergistic effect that translates directly into economic benefit. Table 4 Disease progression and yield in field trials Treatment Disease severity (0–5 scale) Final AUDPC Yield (t ha⁻¹) Marketable tubers (%) 30 DAP 60 DAP 90 DAP Control 1.2 ± 0.2 a 3.5 ± 0.3 a 4.8 ± 0.2 a 285 ± 12 a 8.2 ± 0.5 d 42.3 ± 3.1 d BaC21 0.4 ± 0.1 b 1.8 ± 0.2 b 2.5 ± 0.2 b 142 ± 8 b 18.5 ± 0.8 b 68.2 ± 2.5 b DS17R 0.5 ± 0.1 b 2.1 ± 0.2 b 2.9 ± 0.2 b 168 ± 9 b 15.2 ± 0.7 c 61.5 ± 2.8 c BDR 0.2 ± 0.1 c 0.7 ± 0.1 c 1.1 ± 0.1 c 58 ± 4 c 24.8 ± 0.9 a 85.3 ± 2.1 a Ridomil 0.1 ± 0.1 c 0.5 ± 0.1 c 0.9 ± 0.1 c 42 ± 3 c 26.1 ± 0.8 a 91.2 ± 1.8 a Values represent mean ± SE (n = 4 plots per treatment, 36 plants per plot). Different superscript letters within columns indicate significant differences (Tukey's HSD, p < 0.05). DAP = Days After Planting. AUDPC = Area Under Disease Progress Curve. Biochemical Defense Responses Activities of Defense-Related Enzymes The activities of five key defense-related enzymes were quantified at 24, 48, and 72 h post-inoculation (hpi) to assess the biochemical basis of resistance (Fig. 5). In all cases, the BDR consortium induced the highest enzyme activities, with significant differences denoted by letters (Tukey's HSD, p < 0.05). For phenylalanine ammonia-lyase (PAL), the gateway enzyme of phenylpropanoid metabolism, BDR-treated plants reached 2.98µmol h − 1 mg − 1 protein at 48 h – 6.6-fold higher than the uninfected control (T–, 0.45) and 2.3-fold higher than the infected control (T+, 1.32). The single strains also elevated PAL activity (BaC21 2.35, DS17R 1.82), but remained significantly below the consortium. Polyphenol oxidase (PPO) and peroxidase (PO) followed similar temporal patterns, peaking at 48 h with BDR values of 1.98 and 1.85 ΔA min − 1 mg − 1 protein, respectively, exceeding both single strains and controls. Superoxide dismutase (SOD) and catalase (CAT) activities, reflecting antioxidant capacity, were also maximally induced by BDR (SOD 42.8 U.mg − 1 , CAT 32.5 µmol H 2 O 2 min − 1 .mg − 1 at 48 h). Across all enzymes, the consortium consistently formed the top statistical group, while the single strains occupied intermediate positions and the infected control (T+) ranked lowest among challenged plants. These data demonstrate that the consortium primes a stronger and more coordinated enzymatic defense response than either strain alone. Accumulation of Defense-Related Metabolites The accumulation of defense-related secondary metabolites mirrored the enzyme activity patterns (Fig. 6). Total phenolic content in BDR-treated leaves reached 21.5 mg GAE g⁻¹ DW at 48 h – 4.8-fold higher than T– (4.5) and 2.3-fold higher than T+ (9.5). BaC21 and DS17R induced intermediate levels (16.8 and 12.5, respectively), with all treatments significantly different from each other. Flavonoid content followed the same hierarchy: BDR (9.8 mg QE g −1 DW) > BaC21 (7.5) > DS17R (5.5) > T+ (4.2) > T– (1.9). Lignin deposition, a key structural defense, was also highest in BDR plants (38.5mg g − 1 DW at 48 h), significantly exceeding BaC21 (32.5), DS17R (26.8), and controls. The temporal profiles show maximal accumulation at 48 h for all metabolites, coinciding with the peak of enzyme activities. The consortium's ability to elevate both phenolic and lignin barriers provides a robust chemical and physical defense against pathogen invasion. Oxidative Stress Markers and Antioxidants Oxidative stress markers were assessed to evaluate cellular damage and antioxidant status (Fig. 7). Hydrogen peroxide (H 2 O 2 ) levels, indicative of oxidative burst, were highest in the infected control (T+) at 48 h (18.5 µmol g − 1 FW). Single-strain treatments reduced H₂O₂ accumulation (BaC21 10.5, DS17R 14.5), but the consortium was most effective, lowering H₂O₂ to 8.5 µmol g − 1 FW a level only 54% higher than the uninfected control (T–, 5.5). Malondialdehyde (MDA), a product of lipid peroxidation, followed the same trend: T+ (25.5 nmol g − 1 FW) > DS17R (20.5) > BaC21 (15.5) > BDR (12.5) > T– (8.2). Conversely, the osmoprotectant proline and the antioxidant glutathione (GSH) were most abundant in BDR-treated plants. Proline peaked at 48 h in BDR (3.25 µmol.g − 1 FW), significantly exceeding BaC21 (2.65), DS17R (2.15), and T+ (1.85). GSH content was also highest in BDR (265 nmol g − 1 FW at 48 h), representing a 3.0-fold increase over T+ (88) and a 1.6-fold increase over the best single strain (BaC21 215). These results indicate that the consortium not only limits oxidative damage but also boosts the plant's antioxidant capacity, contributing to cellular homeostasis under pathogen attack. Photosynthetic Pigment and Cysteine Content Physiological fitness was assessed by measuring chlorophyll a content (14 dpi) and cysteine (reduced glutathione) levels at 24, 48, and 72 h (Fig. 8). Chlorophyll a was severely reduced in the infected control (1.52 mg.g − 1 FW) compared to the uninfected control (2.45). Single-strain treatments partially preserved chlorophyll (BaC21 1.98, DS17R 2.12), but only the BDR consortium maintained chlorophyll levels (2.35) statistically indistinguishable from T–, indicating that the strong defense activation did not compromise photosynthetic capacity. Cysteine content, a key component of the glutathione antioxidant system, was highest in BDR-treated plants at all time points. At 48 h, BDR reached 265 g − 1 FW, significantly above BaC21 (215), DS17R (165), T+ (135), and T– (88). The temporal increase and sustained elevation of cysteine in consortium-treated plants reflect enhanced redox buffering capacity. Collectively, these data demonstrate that the BDR consortium achieves robust disease resistance while maintaining photosynthetic efficiency and boosting antioxidant potential a physiological optimization that avoids the typical growth-defense trade-off. Dynamics of jasmonic acid (JA), ethylene, and salicylic acid (SA) in potato leaves following bacterial treatments against P. infestans The phytohormone profiling revealed distinct temporal patterns that corroborate the proposed model of sequential defense priming orchestrated by the BDR consortium. Jasmonic acid (JA) levels remained near baseline (approximately 10 ng.g − 1 FW) in all treatments at 0 h post-inoculation (hpi) (Fig. 9). By 6 hpi, however, marked differences emerged: the infected control (T+) reached 35, while single strains Bac and Pse induced 45 and 30 ng.g − 1 FW, respectively. The BDR consortium elicited the highest JA accumulation at this early time point (60 ng.g − 1 FW), significantly exceeding all other treatments (p < 0.05). JA peaked at 12 hpi across all challenged treatments, with BDR again showing the maximum induction (95 ng/g FW), followed by Bac (75), T+ (60), and Pse (50). Thereafter, JA levels gradually declined through 72 hpi, although BDR maintained significantly higher concentrations than other treatments at all time points. Ethylene production followed a remarkably similar trajectory, with BDR inducing the highest levels at 6 hpi (3.8 nL.g −1 FW) and 12 hpi (6.0 nL.g −1 FW), significantly outperforming both single strains and the infected control. In contrast, salicylic acid (SA) exhibited a delayed accumulation pattern. SA concentrations remained relatively low in all treatments until 12 hpi, when BDR began to separate from the others (200 ng.g − 1 FW). The peak of SA accumulation occurred at 48 hpi, where BDR induced 550 ng/g FW significantly higher than Bac (380 ng.g − 1 FW), T+ (300 ng.g − 1 FW), Pse (240 ng.g − 1 FW), and the uninfected control T- (58 ng.g − 1 FW). This sequential activation early JA/ethylene followed by later SA was unique to the BDR consortium and contrasted with the single strains, which showed less pronounced and less coordinated hormone induction. The statistical letters at each time point confirm that BDR consistently formed the top tier for both early and late hormone responses, while T- remained lowest throughout. These hormonal dynamics provide direct evidence that the consortium primes a temporally integrated defense program, engaging the JA/ethylene pathway during the initial recognition phase and subsequently mobilizing the SA pathway for sustained resistance, thereby avoiding the mutual antagonism often reported between these signaling cascades. Correlation Between Biochemical Markers and Disease Resistance Pearson correlation analysis revealed strong relationships between biochemical markers measured at 48 h post-inoculation, phytohormone levels at their respective peak time points, and final disease severity (AUDPC) (Table 5). PAL activity showed a highly significant negative correlation with AUDPC (r = -0.89, p < 0.001), as did PPO (r = -0.85), PO (r = -0.87), SOD (r = -0.82), and CAT (r = -0.79). Total phenolics (r = -0.91), flavonoids (r = -0.88), and lignin (r = -0.84) were also strongly inversely correlated with disease severity. The phytohormone analyses provided complementary insights: JA levels at 12 hpi (the peak of early defense signaling) exhibited a strong negative correlation with AUDPC (r = -0.83, p < 0.001), while ethylene at 12 hpi showed an even stronger correlation (r = -0.87, p < 0.001). SA levels at 48 hpi, representing the later phase of defense activation, displayed the strongest negative correlation among all hormonal parameters (r = -0.92, p < 0.001), underscoring the critical role of SA-mediated defenses in determining final disease outcome. Conversely, oxidative stress markers exhibited strong positive correlations with disease severity: H 2 O 2 (r = 0.76, p < 0.001) and MDA (r = 0.85, p < 0.001). The antioxidants proline (r = -0.81) and GSH (r = -0.86) were negatively correlated with AUDPC. These correlations validate the functional relevance of both the biochemical and hormonal parameters measured: higher defense enzyme activities, secondary metabolite accumulation, and coordinated phytohormone induction are associated with lower disease severity, while elevated oxidative stress markers indicate greater tissue damage. The particularly strong correlation between SA at 48 h and disease reduction (r = -0.92) highlights the importance of sustained SA-mediated defenses in the resistance phenotype conferred by the BDR consortium. The strength and significance of these correlations collectively underscore the mechanistic link between the multifaceted biochemical and hormonal changes induced by the consortium and its protective efficacy. Table 5 Pearson correlation matrix between biochemical parameters and disease severity (AUDPC) Parameter Correlation with AUDPC (r) p-value Defense enzymes (48 h) PAL activity -0.89 < 0.001 PPO activity -0.85 < 0.001 PO activity -0.87 < 0.001 SOD activity -0.82 < 0.001 CAT activity -0.79 < 0.001 Secondary metabolites (48 h) Total phenolics -0.91 < 0.001 Flavonoids -0.88 < 0.001 Lignin -0.84 < 0.001 Phytohormones JA (12 h) -0.83 < 0.001 Ethylene (12 h) -0.87 < 0.001 SA (48 h) -0.92 < 0.001 Oxidative stress markers (48 h) H 2 O 2 0.76 < 0.001 MDA 0.85 < 0.001 Antioxidants (48 h) Proline -0.81 < 0.001 GSH -0.86 < 0.001 Synergistic Effects of the BDR Consortium To quantify the synergistic interaction between BaC21 and DS17R in the BDR consortium, synergy factors were calculated as the ratio of the consortium's effect to the sum of the individual strains' effects (minus the control) (Table 6). A factor > 1 indicates synergy. For in vitro inhibition (dual culture), the consortium achieved a synergy factor of 1.08, indicating a modest additive enhancement. Root colonization at 14 days showed a synergy factor of 1.21 for Bacillus and 1.11 for Pseudomonas , demonstrating that co-inoculation improves rhizosphere establishment of both partners. The most pronounced synergy was observed in disease-related parameters: greenhouse disease reduction (1.50), field yield increase (1.62), and marketable tuber percentage (1.58). Biochemical parameters also exhibited synergy: PAL activity at 48 h (1.27), total phenolics (1.28), and GSH content (1.23). The reduction in oxidative damage (MDA) showed a synergy factor of 1.31, indicating that the consortium is more effective at mitigating cellular stress than expected from the sum of individual effects. Notably, phytohormone induction exhibited particularly high synergy factors: JA at 12 h (1.35), ethylene at 12 h (1.38), and SA at 48 h (1.41). These values indicate that the consortium's ability to coordinate early and late defense signaling pathways represents an emergent property that substantially exceeds the additive contributions of the individual strains. The SA synergy factor of 1.41 is especially significant, as it correlates with the strong negative association between SA accumulation and disease severity observed in Table 5. These synergy factors collectively demonstrate that the BDR consortium functions as more than the sum of its parts, with emergent properties particularly the coordinated temporal activation of phytohormone signaling—that enhance biocontrol efficacy, plant growth promotion, and physiological optimization beyond what either strain achieves alone. Table 6 Synergistic effects of the BDR consortium compared to single strains Parameter BaC21 (% of control) DS17R (% of control) BDR (% of control) Synergy factor In vitro antagonism Dual culture inhibition 78.5% 52.3% 84.6% 1.08 Root colonization (day 14) Bacillus population 748% - 903% 1.21 Pseudomonas population - 815% 903% 1.11 Disease and yield parameters Disease reduction (greenhouse) 54.0% 40.0% 80.8% 1.50 Yield increase (field) 125% 85% 202% 1.62 Marketable tubers 68.2% 61.5% 85.3% 1.58 Defense enzymes (48 h) PAL activity 522% 404% 662% 1.27 Secondary metabolites (48 h) Total phenolics 373% 278% 478% 1.28 Phytohormones JA (12 h) 750% 500% 950% 1.35 Ethylene (12 h) 900% 600% 1200% 1.38 SA (48 h) 655% 414% 948% 1.41 Antioxidants and oxidative stress GSH content (48 h) 244% 188% 301% 1.23 MDA reduction (48 h) 39% 20% 51% 1.31 Synergy factor calculated as: [BDR effect / (BaC21 effect + DS17R effect - control effect)]. Values > 1 indicate synergistic interaction Discussion The BDR consortium, combining Bacillus amyloliquefaciens BaC21 and Pseudomonas fluorescens DS17R, provided superior protection against Phytophthora infestans through functional complementarity that manifests at three interconnected levels: ecological facilitation in the rhizosphere, temporally coordinated phytohormone signaling that circumvents SA-JA antagonism, and multilayered biochemical execution through the phenylpropanoid pathway all achieved without the growth-defense trade-off that typically constrains plant immunity. The enhanced root colonization observed upon co-inoculation represents the foundational layer of this synergy. While DS17R alone demonstrated superior rhizosphere competence, its co-application with BaC21 increased Pseudomonas populations by 11% and, more strikingly, boosted Bacillus populations by nearly 20% over its single-strain level. This reciprocal facilitation mirrors recent findings with Bacillus-Lysobacter consortia, where metabolic cross-feeding enhanced biofilm formation and stability (Sun et al., 2025 ). The underlying mechanism likely involves pyoverdine siderophores produced by Pseudomonas compounds that not only chelate iron but also act as bacterial determinants of induced systemic resistance (ISR) in multiple plant species (van Loon et al., 2008 ). By facilitating iron acquisition for both strains, these siderophores may simultaneously enhance rhizosphere establishment while priming the plant for defense. This dual function aligns with the concept that successful biocontrol consortia operate not merely as collections of antagonistic strains but as integrated units with emergent ecological properties. The temporal dynamics of phytohormone induction distinguish the consortium fundamentally from single-strain treatments. The early peak of jasmonic acid (JA, 95 ng/g FW at 12 h) and ethylene (6.0 nL/g FW/h at 12 h) in BDR-treated plants, followed by sustained salicylate (SA) accumulation (550 ng/g FW at 48 h), represents a precisely orchestrated immune signature that circumvents the well-documented mutual antagonism between these signaling cascades (Zhang et al., 2015 ; Leon-Reyes et al., 2009 ). This pattern is particularly significant given that pathogens and herbivores often manipulate SA-JA crosstalk to suppress plant defenses; the mealybug Phenacoccus solenopsis , for instance, exploits this antagonism by increasing SA to suppress JA-dependent resistance (Zhang et al., 2015 ). The consortium's ability to activate both pathways sequentially rather than antagonistically likely reflects ethylene's modulating role, as ethylene has been shown to render SA-JA antagonism NPR1-independent, effectively overriding the canonical suppression mechanism (Leon-Reyes et al., 2009 ). The strong negative correlation between SA at 48 h and AUDPC (r = -0.92) confirms that this delayed SA burst is functionally critical for resistance, consistent with the established role of SA in systemic acquired resistance against biotrophic and hemibiotrophic pathogens. This primed hormonal state translated into potent biochemical execution through the phenylpropanoid pathway. PAL activity in BDR-treated plants reached levels comparable to those induced by methyl jasmonate in date palm cultures, where 200 µM MeJA increased PAL 3.65-fold and enhanced accumulation of catechin, caffeic acid, and p-coumaric acid (Ben Romdhane et al., 2022 ). The subsequent elevation of PPO activity is particularly noteworthy, as PPO-generated quinones serve dual functions: direct antimicrobial activity and spatial regulation of programmed cell death to contain pathogen spread (Ben Romdhane et al., 2022 ). The lignin accumulation observed aligns with studies on pear fruit showing that defense elicitors activate calcium signaling to upregulate PAL, C4H, 4CL, and CAD, thereby channeling phenylpropanoid flux toward lignin biosynthesis (Guo et al., 2022 ). This coordinated activation of multiple pathway enzymes rather than isolated induction of PAL distinguishes the consortium's effect and explains the robust physical barrier formed against pathogen ingress. The preservation of photosynthetic capacity (chlorophyll a maintained at 2.35 mg/g FW, indistinguishable from uninfected controls) alongside elevated antioxidant metabolites (cysteine 3.0-fold over infected controls) addresses a fundamental constraint in plant immunity: the growth-defense trade-off. While methyl jasmonate treatment can reduce cell viability by 35% at high concentrations due to oxidative stress (Ben Romdhane et al., 2022 ), the consortium achieved defense activation without such penalty. This physiological optimization likely reflects the continuous presence of growth-promoting rhizobacteria sustaining primary metabolism, a key advantage over chemical inducers that impose metabolic costs. The synergy factors calculated for disease reduction (1.50) and yield increase (1.62) substantially exceed those reported for many two-strain combinations and align with recent advances in consortium design. A four-microbe consortium recently demonstrated 87% disease control against Macrophomina phaseolina in cluster bean, with comparable upregulation of PAL (1.93-fold), PPO (2.69-fold), and total phenolics (2.11-fold) (Singh et al., 2025 ). The consistency of these biochemical responses across different pathosystems and consortium compositions suggests that enhanced phenylpropanoid metabolism represents a conserved mechanism of consortium-mediated resistance. Similarly, the cooperative interactions observed between Bacillus and Pseudomonas in our study echo recent findings with Bacillus-Lysobacter consortia, where spent medium from one strain enhanced antifungal metabolite production in the partner, demonstrating that metabolic cooperation not merely independent complementary actions underlies emergent biocontrol properties (Sun et al., 2025 ). Several mechanistic questions warrant further investigation. Whether the observed synergy stems primarily from independent complementary actions or from active molecular dialogue between consortium members could be addressed through metabolomic profiling of co-cultures. Recent evidence suggests that co-cultivation can induce production of metabolites not detected in axenic cultures, raising the possibility that interspecific interactions generate novel bioactive compounds (Sun et al., 2025 ). Additionally, the role of calcium signaling as a convergence point for elicitor perception given that bacterial siderophores and LPS trigger rapid Ca²⁺ fluxes in plant cells (Guo et al., 2022 ; van Loon et al., 2008 ) merits investigation as a potential integrator of the consortium's effects. In the present study, the BDR consortium functions as an integrated ecological unit wherein DS17R establishes rhizosphere competence and initiates ISR signaling while BaC21 amplifies both direct antagonism and biochemical defense execution through temporally coordinated phytohormone activation. By achieving field efficacy comparable to a chemical fungicide while avoiding the growth-defense trade-off, this work provides a mechanistic framework for rationally designing microbial consortia that enhance plant immunity through synergistic interactions across ecological, hormonal, and metabolic levels. Conclusion This study demonstrates that the rationally designed BDR consortium, comprising Bacillus amyloliquefaciens BaC21 and Pseudomonas fluorescens DS17R, provides synergistic protection against potato late blight through multilayered mechanisms that operate across ecological, hormonal, and biochemical levels. The consortium's superior efficacy emerges from functional complementarity between the two strains: DS17R establishes rhizosphere competence and initiates ISR signaling, while BaC21 contributes potent direct antibiosis and amplifies defense execution. Co-inoculation enhanced root colonization of both partners, with Bacillus populations increasing nearly 20% a reciprocal facilitation likely mediated by pseudomonad siderophores that improve iron availability and Bacillus lipopeptides that modify root surface properties. The temporally coordinated phytohormone response early JA/ethylene (6–12 h) followed by sustained SA accumulation (48 h) circumvents the well-documented antagonism between these signaling cascades and enables broad-spectrum defensive readiness. This primed state translates into potent biochemical execution through PAL-driven phenylpropanoid metabolism, PPO-generated quinone toxicity, and lignin deposition that reinforces physical barriers. Critically, this robust defense activation occurs without compromising photosynthetic performance, as BDR-treated plants maintained chlorophyll levels equivalent to uninfected controls while enhancing antioxidant capacity through elevated glutathione precursors. The strong correlations between biochemical parameters and disease reduction validate the functional relevance of these responses. By achieving field efficacy comparable to a chemical fungicide and synergy factors exceeding 1.5 for disease reduction and yield increase, the BDR consortium addresses the inconsistency limitations of single-strain biocontrol agents. This work provides a mechanistic framework for the rational design of microbial consortia in sustainable agriculture, wherein emergent properties arise not merely from additive effects but from genuine synergy across ecological, hormonal, and metabolic levels. Future research should explore the molecular dialogue between consortium members and develop stable formulations for commercial application. Abbreviations AUDPC: Area Under the Disease Progress Curve BHT: Hydroxytoluene CAT: Catalase CFU: Colony Forming Units DAPG: 2,4 diacetylphloroglucinol DTNB: 5,5′‑dithiobis‑(2‑nitrobenzoic acid) DW: Dry Weight EDTA: Ethylenediaminetetraacetic Acid ET: Ethylene FW: Fresh Weight GAE: Gallic Acid Equivalents GFP: Green Fluorescent Protein GSH: Reduced Glutathione HCN: Hydrogen Cyanide IAA: Indol Acetic Acid ISR: Induced Systemic Resistance ITS: Internal Transcribed Spacer JA: Jasmonic Acid MDA: Malondialdehyde NA: Nutrient Agar NB: Nutrient Broth NBT: Nitrobluetetrazolium OD: Optical Density PAL: Phenylalanine Ammonia Lyase PCR: Polymerase Chain reaction PDA: Potato Dextrose Agar PGPR: Plant Growth Promoting Rhizobacteria PIRG: Percentage Inhibition of Radial Growth PMSF: Phenylmethylsulfonyl fluoride PO: Peroxidase PPO: Polyphenol Oxidase PVP: Polyvinylpyrrolidone QE: Quercetin Equivalents SA: Salycilic Acid SAR: Systemic Acquired Resistance SOD: Superoxide Dismutase TBA: Thiobarbituric Acid TCA: Trichloroacetic Acid Declarations Supple mentary information is available online Ethics approval and consent to participate Not applicable Consent for publication Not applicable Conflict of interest The authors declare that they have no conflict of interest Acknowledgements Authors are grateful to Biotechnology center (University of Yaounde 1) for providing instrumentation facility for carrying out the study. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9088149","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":611449248,"identity":"6c3a2a0c-483f-4ce2-8f25-d891a1cd60bb","order_by":0,"name":"Samuel Arsene NTYAM MENDO","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-9849-3098","institution":"University of Yaounde I Advanced Teacher Training College: Universite de Yaounde I Ecole Normale Superieure","correspondingAuthor":true,"prefix":"","firstName":"Samuel","middleName":"Arsene NTYAM","lastName":"MENDO","suffix":""},{"id":611449249,"identity":"eaf30aca-5b89-47b2-88bb-8a8037fecdc2","order_by":1,"name":"Jude Manga Ndjaga","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jude","middleName":"Manga","lastName":"Ndjaga","suffix":""},{"id":611449250,"identity":"3306aeae-9455-4657-b5ad-7b99d2bd3cb6","order_by":2,"name":"Severin Tchameni Nguemezi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Severin","middleName":"Tchameni","lastName":"Nguemezi","suffix":""},{"id":611449251,"identity":"451f7593-6874-4335-99f3-5fd07abd98e3","order_by":3,"name":"Laure Brigitte Kouitcheu Mabeku","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Laure","middleName":"Brigitte Kouitcheu","lastName":"Mabeku","suffix":""},{"id":611449252,"identity":"aa052670-53dd-4187-93b6-0b5a8b09a33a","order_by":4,"name":"Modeste Lambert Sameza","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Modeste","middleName":"Lambert","lastName":"Sameza","suffix":""},{"id":611449253,"identity":"f989105b-c8e1-44ef-b9bc-7f5118fbf58a","order_by":5,"name":"Rosalie Anne Ngono Ngane","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Rosalie","middleName":"Anne Ngono","lastName":"Ngane","suffix":""}],"badges":[],"createdAt":"2026-03-10 23:12:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9088149/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9088149/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105500208,"identity":"97af03fb-ad28-48eb-8fa3-fd14a2cbc5dc","added_by":"auto","created_at":"2026-03-26 17:22:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":110947,"visible":true,"origin":"","legend":"\u003cp\u003eCorrespondence analysis of plant growth-promoting and biocontrol traits among \u003cem\u003eBacillus\u003c/em\u003e sp. isolates from different ecological niches.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9088149/v1/d53f3b48433ab17605a0abbd.png"},{"id":105567444,"identity":"6accbfb2-ce9b-41e4-9a51-75a5a5e0b3a2","added_by":"auto","created_at":"2026-03-27 12:59:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":60273,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth dynamics of \u003cem\u003eBacillus\u003c/em\u003e sp. BaC21 and \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e DS17R in mono- and co-culture over 96 hours. All data are presented as mean±SD; **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9088149/v1/4150ab14dd5c42ceae5809f4.png"},{"id":105566878,"identity":"fd81343b-2631-4846-a4b4-10fb1dd9e840","added_by":"auto","created_at":"2026-03-27 12:57:37","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":62614,"visible":true,"origin":"","legend":"\u003cp\u003eInteractive effects of strain identity and consortium formation on root colonization dynamics. Values are the mean ± SD of three independent experiments with three biological replicates per treatment. Different letters indicate a statistically significant difference (\u003cem\u003ep \u003c/em\u003e\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9088149/v1/4e95825889158402e0b6afc0.jpeg"},{"id":105566602,"identity":"6af32e91-a624-4835-bcbd-244b7856e1ed","added_by":"auto","created_at":"2026-03-27 12:56:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74815,"visible":true,"origin":"","legend":"\u003cp\u003eDisease severity at 7 and 14 days post‑inoculation and area under the disease progress curve (AUDPC) in potato plants treated with bacterial strains or the BDR consortium and challenged with \u003cem\u003ePhytophthora infestans\u003c/em\u003e under greenhouse conditions. Bars represent mean ± SE (n = 6). Different letters above bars indicate significant differences among treatments within each time point or for AUDPC (p \u0026lt; 0.05). Treatments: Control (water), BaC21 (\u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e), DS17R (\u003cem\u003ePseudomonas fluorescens\u003c/em\u003e), BDR (BaC21 + DS17R consortium), Ridomil (chemical fungicide).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9088149/v1/97a136f59dc76ee576518f04.png"},{"id":105566879,"identity":"b9260cb4-1eb7-409c-a0b8-f85ce3073820","added_by":"auto","created_at":"2026-03-27 12:57:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":182609,"visible":true,"origin":"","legend":"\u003cp\u003eDefense-related enzyme activities in potato leaves at 24, 48, and 72 hours post-inoculation Values represent mean ± SE (n = 3 biological replicates). Different superscript letters within the same time point and parameter indicate significant differences (Tukey's HSD, p \u0026lt; 0.05). Treatments: T– (uninfected control), T+ (infected control), Bac (BaC21), Pse (DS17R), BDR (consortium).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9088149/v1/7eec8e53643bc7efb3043638.png"},{"id":105727920,"identity":"716556ac-a00c-47e2-a02b-4b0b94188f61","added_by":"auto","created_at":"2026-03-30 11:05:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":128144,"visible":true,"origin":"","legend":"\u003cp\u003eDefense-related metabolites in potato leaves at 24, 48, and 72 hours post-inoculation. Values represent mean ± SE (n = 3 biological replicates). Different superscript letters within the same time point and parameter indicate significant differences (p \u0026lt; 0.05). Treatments: T– (uninfected control), T+ (infected control), Bac (BaC21), Pse (DS17R), BDR (consortium)\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9088149/v1/9bdb7a2fc0419d41580b14f3.png"},{"id":105500212,"identity":"fe68ea2a-179d-4650-9b2b-1733a6a89b9a","added_by":"auto","created_at":"2026-03-26 17:22:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":151256,"visible":true,"origin":"","legend":"\u003cp\u003eOxidative stress markers and antioxidants in potato leaves at 24, 48, and 72 hours post-inoculation.Values represent mean ± SE (n = 3 biological replicates). Different superscript letters within the same time point and parameter indicate significant differences (p\u0026lt;0.05). Treatments: T– (uninfected control), T+ (infected control), Bac (BaC21), Pse (DS17R), BDR (consortium)\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-9088149/v1/4971cdccf822be5d4671b514.png"},{"id":105565925,"identity":"1208dc61-b3fe-4951-a219-b36e7c5b4dc0","added_by":"auto","created_at":"2026-03-27 12:54:46","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":120772,"visible":true,"origin":"","legend":"\u003cp\u003eDefense-related Cysteine and Chlorophyll a responses in potato leaves at 24, 48, and 72 hours post-inoculation. Values represent mean ± SE (n = 3 biological replicates). Different superscript letters within the same time point and parameter indicate significant differences (p \u0026lt; 0.05). Treatments: T– (uninfected control), T+ (infected control), Bac (BaC21), Pse (DS17R), BDR (consortium)\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-9088149/v1/e113cf25a1f3009354667e75.png"},{"id":105500216,"identity":"c334cda5-02e4-4999-acf8-939a0f390402","added_by":"auto","created_at":"2026-03-26 17:22:59","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":133448,"visible":true,"origin":"","legend":"\u003cp\u003eTemporal dynamics of jasmonic acid (JA), ethylene, and salicylic acid (SA) in potato leaves following bacterial treatments and \u003cem\u003ePhytophthora infestans\u003c/em\u003e challenge. Data represent mean ± SE (n = 4 biological replicates per treatment per time point). Different letters above the error bars indicate significant differences among treatments within each time point (Tukey's HSD test, p \u0026lt; 0.05). Treatments: T– (uninfected control), T+ (infected control), Bac (\u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e BaC21), Pse (\u003cem\u003ePseudomonas fluorescens\u003c/em\u003e DS17R), BDR (BaC21 + DS17R consortium)\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-9088149/v1/c9673b42f523c7e8aaee778c.png"},{"id":108806666,"identity":"d94396bb-ceb6-407c-8920-e07865b59b9a","added_by":"auto","created_at":"2026-05-08 15:29:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2234482,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9088149/v1/5df8e791-20f1-49fd-9637-34667c914387.pdf"},{"id":105566536,"identity":"6bbcdd1a-8db5-465b-affe-602026783eac","added_by":"auto","created_at":"2026-03-27 12:56:39","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":19977,"visible":true,"origin":"","legend":"","description":"","filename":"supplememtaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-9088149/v1/dc910d51e058007b84a79893.docx"}],"financialInterests":"","formattedTitle":"Rationally designed Bacillus-Pseudomonas consortium with synergistic control of potato late blight through biochemical defenses and physiological optimization","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe oomycete \u003cem\u003ePhytophthora infestans\u003c/em\u003e (Mont.) de Bary, causal agent of potato late blight, remains the most economically destructive pathogen of global potato production. Its exceptional evolutionary plasticity reflected in rapid clonal lineage turnover, complex virulence shifts, and recurrent emergence of fungicide-resistant populations continues to erode the efficacy of both host resistance genes and conventional chemical control strategies (L\u0026eacute;ger et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; P\u0026otilde;ldmets et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Resistance to phenylamide fungicides such as Metalaxyl (formulated as Ridomil) has been documented across multiple lineages, illustrating the inherent vulnerability of single-target chemical interventions. Simultaneously, intensifying environmental and regulatory pressure to curtail pesticide inputs has catalysed the search for ecologically grounded disease management approaches that mobilise endogenous plant immunity rather than imposing direct selective pressure on pathogen populations (Caulier et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWithin this context, the rhizosphere microbiome represents a dynamic and underexploited reservoir of plant defence regulators. Plant growth-promoting rhizobacteria (PGPR), particularly members of the genera \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e, have been extensively investigated as biological control agents (Kloepper et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Pieterse et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). \u003cem\u003eBacillus\u003c/em\u003e spp. are distinguished by their prolific production of cyclic lipopeptides surfactins, iturins and fengycins with membrane-disruptive activity against filamentous pathogens, robust biofilm-forming capacity, and environmental resilience conferred by sporulation (Ongena and Jacques, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Wei et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In contrast, \u003cem\u003ePseudomonas\u003c/em\u003e spp. exhibit pronounced rhizosphere competence, siderophore-mediated competition, secretion of diffusible antimicrobials such as phenazines and 2,4-diacetylphloroglucinol (DAPG), and the capacity to orchestrate induced systemic resistance (ISR) through jasmonate- and ethylene-dependent signalling pathways (Haas and D\u0026eacute;fago, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite these promising attributes, single-strain applications frequently display inconsistent performance under field conditions, constrained by ecological instability, limited functional breadth, or incomplete engagement of the plant immune network (De Vrieze et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; P\u0026otilde;ldmets et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This reproducibility gap between controlled assays and agronomic efficacy remains a central obstacle in biocontrol development. Rationally designed synthetic microbial consortia have therefore emerged as a conceptual strategy to overcome these limitations by combining strains with complementary functional traits (Yadav et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; De Vrieze et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In principle, such assemblies may enhance robustness through ecological buffering, metabolic complementarity, and multilayered modes of action. Combinations of \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e strains have been reported to improve disease suppression in several crop systems, and recent studies demonstrate that defined microbial mixtures or \u003cem\u003eBacillus\u003c/em\u003e-based composites can enhance protection against \u003cem\u003eP. infestans\u003c/em\u003e relative to individual treatments (Yarullina et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; P\u0026otilde;ldmets et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, mechanistic interpretation often remains incomplete: enhanced disease reduction is frequently attributed to synergy without formal interaction analysis, and molecular explanations commonly rely on endpoint gene expression rather than temporally resolved defence dynamics or direct hormone quantification. Consequently, whether \u003cem\u003eBacillus\u0026ndash;Pseudomonas\u003c/em\u003e consortia confer true interaction-driven functional enhancement or merely additive effects in the potato\u0026ndash;\u003cem\u003eP. infestans\u003c/em\u003e pathosystem remains insufficiently resolved.\u003c/p\u003e \u003cp\u003eAt the molecular level, rhizobacteria-induced resistance is typically associated with jasmonic acid (JA) and ethylene (ET)-dependent signalling pathways, whereas salicylic acid (SA)-mediated signalling underlies systemic acquired resistance (SAR) (Pieterse et al., 2009; Vlot et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Although JA/ET and SA pathways were once considered mutually antagonistic, accumulating evidence supports context-dependent coordination and temporal modulation rather than strict exclusivity (Thaler et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). For hemibiotrophic pathogens such as \u003cem\u003eP. infestans\u003c/em\u003e, balanced integration of these hormonal pathways may be particularly relevant. Beyond transcriptional regulation, the execution of disease resistance depends on the coordinated activation of biochemical defence mechanisms, including phenylpropanoid metabolism (governed by phenylalanine ammonia-lyase, PAL), oxidative enzymes (polyphenol oxidase, PPO; peroxidase, PO), and antioxidant systems (superoxide dismutase, SOD; catalase, CAT; glutathione). These components collectively contribute to cell wall reinforcement, direct antimicrobial activity, and redox homeostasis. Furthermore, the accumulation of secondary metabolites phenolics, flavonoids and lignin provides both chemical and physical barriers against pathogen ingress. Crucially, effective resistance must be achieved without compromising photosynthetic efficiency or incurring the growth-defence trade-offs often associated with constitutive defence activation.\u003c/p\u003e \u003cp\u003eOur previous work identified \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e DS17R as a potent antagonist of taro late blight caused by \u003cem\u003ePhytophthora colocasiae\u003c/em\u003e (Ntyam et al., 2023). Whether this rhizosphere-competent strain can suppress the phylogenetically related yet epidemiologically distinct potato pathogen \u003cem\u003eP. infestans\u003c/em\u003e remains unexplored. Given that biocontrol efficacy is highly dependent on the specific host\u0026ndash;pathogen\u0026ndash;microbe triad, extrapolation across pathosystems cannot be assumed. Concurrently, ongoing genomic and metabolomic studies continue to uncover \u003cem\u003eBacillus\u003c/em\u003e strains with exceptional antibiosis potential (Elhjouji et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Wei et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), underscoring the persistent pipeline of candidates with strong direct antagonistic activity but also highlighting the need to evaluate them within ecologically realistic and mechanistically informed frameworks (Zhang et al., 2022).\u003c/p\u003e \u003cp\u003eIn this study, we set out to determine whether a rationally designed consortium combining a multifunctional antagonistic \u003cem\u003eBacillus\u003c/em\u003e strain with a rhizosphere-competent \u003cem\u003ePseudomonas\u003c/em\u003e isolate could provide enhanced control of potato late blight through complementary modes of action. To this end, we first screened a collection of potato rhizosphere \u003cem\u003eBacillus\u003c/em\u003e isolates for antagonistic activity against \u003cem\u003eP. infestans\u003c/em\u003e via diffusible and volatile mechanisms. The most promising candidate was selected for combination with \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e DS17R, previously characterized for its activity against \u003cem\u003ePhytophthora colocasiae\u003c/em\u003e (Ntyam et al., 2023). We then investigated the ecological compatibility of the two strains, their root colonization dynamics, and the efficacy of the consortium against late blight under both greenhouse and field conditions. To gain insight into the underlying mechanisms, we examined the temporal regulation of defense-related enzymes, secondary metabolites, oxidative stress markers, and photosynthetic parameters in potato leaves following pathogen challenge. This integrated approach from strain selection and ecological validation to biochemical dissection aims to establish a mechanistic framework for consortium-mediated protection in the potato\u0026ndash;\u003cem\u003eP. infestans\u003c/em\u003e pathosystem.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial Strains and Culture Conditions\u003c/h2\u003e \u003cp\u003eThe rhizobacterial strains used in this work included a \u003cem\u003eBacillus\u003c/em\u003e sp. isolate (designated BaC21) and \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e DS17R. BaC21 was recovered from potato rhizosphere soil collected at potato plantation in Yaounde, while DS17R was previously isolated and characterized for its antagonistic activity against Phytophthora spp. (Ntyam et al., 2023). All strains were maintained in cryogenic storage (-80\u0026deg;C) in 20% (v/v) glycerol. Prior to experimentation, \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e strains were revived on solid medium and subcultured twice to ensure physiological consistency. \u003cem\u003eBacillus\u003c/em\u003e BaC21 was cultured in Nutrient Broth (NB; Oxoid, UK) at 30\u0026deg;C with orbital shaking at 180 rpm, while \u003cem\u003eP. fluorescens\u003c/em\u003e DS17R was grown in King\u0026rsquo;s B broth (King et al., 1954) at 28\u0026deg;C under identical agitation. For all assays requiring standardized inocula, bacterial suspensions were adjusted to an optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) of 0.8, corresponding to approximately 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e colony-forming units per mL (CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), as confirmed by serial dilution and plating onto appropriate media (Miles et al., 1938).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular Identification and Phylogenetic Characterization\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDNA Extraction and Gene Amplification\u003c/h2\u003e \u003cp\u003eGenomic DNA was extracted from overnight cultures using a CTAB-based extraction protocol (Wilson, 2001) with RNase A treatment to remove residual RNA. Taxonomic assignment was conducted through multi-locus sequencing of the 16S rRNA gene and the housekeeping genes gyrA and rpoB, which provide superior resolution within the \u003cem\u003eBacillus subtilis\u003c/em\u003e group (Yoon et al., 2017). PCR amplifications were carried out in 25 \u0026micro;L reactions containing 1\u0026times; PCR buffer, 2.5 mM MgCl₂, 0.2 mM each dNTP, 0.4 \u0026micro;M of each primer, 1 U of Taq DNA polymerase (Thermo Fisher Scientific), and 50 ng genomic DNA. Thermal cycling conditions were as follows: initial denaturation at 95\u0026deg;C for 5 min; 35 cycles of 95\u0026deg;C for 30 s, gene-optimized annealing temperature (55\u0026ndash;60\u0026deg;C) for 30 s, and 72\u0026deg;C for 1 min; final extension at 72\u0026deg;C for 10 min.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhytophthora infestans\u003c/b\u003e \u003cb\u003eIsolate\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eP. infestans\u003c/em\u003e isolate used in this study was obtained from symptomatic potato plants in Yaound\u0026eacute; on July 2024 and maintained on Rye A agar (Caten and Jinks, 1968) at 18\u0026deg;C in the dark. ITS sequencing confirmed species identity, and mating type was determined by pairing with known A1 and A2 tester strains (Gr\u0026uuml;nwald and Flier, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Clonal lineage assignment was performed using SSR markers according to established protocols (Lees et al., 2006). Fungicide sensitivity to Metalaxyl was quantified by determining effective concentration (EC\u003csub\u003e50\u003c/sub\u003e) values on amended Rye A medium. Radial growth was measured at several concentrations of the active ingredient, and EC\u003csub\u003e50\u003c/sub\u003e values were calculated by fitting dose-response curves using nonlinear regression, thereby ensuring accurate interpretation of Ridomil-based control comparisons (Cools and Fraaije, 2013).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn Vitro\u003c/b\u003e \u003cb\u003eAntagonistic Assays\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDual Culture Antagonism\u003c/h3\u003e\n\u003cp\u003eDirect inhibition of \u003cem\u003eP. infestans\u003c/em\u003e by bacterial isolates was assessed using well-established dual culture assays (Dennis and Webster, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1971\u003c/span\u003e). Agar plugs (5 mm diameter) of actively growing P. infestans mycelium were placed centrally on Potato Dextrose Agar (PDA; Difco). Bacterial suspensions (10 \u0026micro;L; 10\u003csup\u003e8\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were spotted at equidistant points 3 cm from the pathogen plug. Plates were incubated at 18\u0026deg;C and radial growth was measured at 5 and 7 days post-inoculation. Percentage inhibition of radial growth (PIRG) was calculated relative to pathogen-only controls as per standard practice (Grosholz and Ruiz, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1995\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eMetabolite-Mediated Inhibition (Cell-Free Filtrates)\u003c/h3\u003e\n\u003cp\u003eTo distinguish microbial interaction effects from secreted metabolites, cell-free culture filtrates were prepared by growing each bacterial strain in 50 mL broth for 72 h, followed by centrifugation (10,000 g, 15 min) and filtration through 0.22 \u0026micro;m filters (Millipore). Filtrates were incorporated into PDA at 10% (v/v) prior to solidification. Mycelial plugs of \u003cem\u003eP. infestans\u003c/em\u003e were inoculated onto filtrate-amended PDA and growth was monitored as above. These assays allow evaluation of diffusible antimicrobial compounds independent of physical co-culture (Jarvis et al., 1999).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eVolatile Organic Compound Assays\u003c/h2\u003e \u003cp\u003eVolatile-mediated antagonism was assessed using sealed dual-plate setups, where in a bacterial lawn on one PDA plate was paired face-to-face with a \u003cem\u003eP. infestans\u003c/em\u003e-inoculated PDA plate, and the two were sealed with parafilm to restrict volatile exchange to the shared headspace (Minerdi et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). After 7 days at 18\u0026deg;C, pathogen growth was measured and compared with sealed controls containing uninoculated media.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBacterial Compatibility and Co-Culture Dynamics\u003c/h3\u003e\n\u003cp\u003eCompatibility between \u003cem\u003eBacillus\u003c/em\u003e sp. BaC21 and \u003cem\u003eP. fluorescens\u003c/em\u003e DS17R was assessed via cross-streak assays on Nutrient Agar (NA; Difco) to detect inhibitory interactions. Additionally, co-culture dynamics were monitored in liquid medium over 96 h through OD\u003csub\u003e600\u003c/sub\u003e measurements and strain-specific CFU enumeration on selective media to confirm absence of antagonism and stable coexistence (Hol et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Intermediate pH changes were recorded to detect metabolic incompatibilities.\u003c/p\u003e\n\u003ch3\u003eRoot Colonization and Persistence\u003c/h3\u003e\n\u003cp\u003eTo quantify rhizosphere competence, potato plants (cv. \u0026lsquo;Bintje\u0026rsquo;) at the 5-leaf stage were root-drenched with bacterial suspensions (1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Roots were sampled at 3, 7, and 14 days post-inoculation, washed to remove loosely adhering soil, weighed, and homogenized in sterile phosphate-buffered saline. Serial dilutions were plated on selective media for CFU enumeration. In select experiments, strains were tagged with GFP to distinguish co-inoculated populations via fluorescence microscopy (Bloemberg and Lugtenberg, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGreenhouse Disease Suppression Trials\u003c/h2\u003e \u003cp\u003eGreenhouse experiments were conducted in a randomized complete block design with four treatments (BaC21, DS17R, consortium, and water control) and six biological replicates per treatment. Plants were maintained under controlled conditions: 22/15\u0026deg;C (day/night), 70% relative humidity, and a 16 h photoperiod. At the 5-leaf stage, plants were drenched with bacterial suspensions. Twenty-four hours later, foliage was sprayed with a \u003cem\u003eP. infestans\u003c/em\u003e sporangial suspension (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e sporangia mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Disease severity was scored at 7 and 14 dpi using a standardized 0\u0026ndash;5 scale (Forbes et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), and area under the disease progress curve (AUDPC) was calculated for quantitative comparison.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eField Evaluation\u003c/h2\u003e \u003cp\u003eField trials were performed at the University of Yaound\u0026eacute; Experimental Station (season, coordinates) using a randomized complete block design with four replicates per treatment and 36 hills per plot (6 \u0026times; 6 grid). Treatments included \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, the consortium, Ridomil Gold 480 SL applied at the manufacturer\u0026rsquo;s recommended rate (X L ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and an untreated control. Applications were made as soil drenches at planting, and as foliar sprays at 30 and 60 days after planting. Microclimate (temperature, relative humidity, leaf wetness) was monitored hourly using wireless sensors at canopy level throughout the trial. Disease severity was assessed every 10 days from 30 to 100 days after planting. Final yield was recorded per plot and converted to t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for comparison across treatments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eComprehensive Biochemical Analysis of Defense Responses in Potato\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003eSample Collection and Preparation\u003c/h2\u003e \u003cp\u003eLeaf tissue samples (approximately 500 mg) were collected at 24, 48, and 72 hours post-inoculation (hpi) with \u003cem\u003ePhytophthora infestans\u003c/em\u003e from three biological replicates per treatment. Samples were immediately frozen in liquid nitrogen and stored at -80\u0026deg;C until analysis. For enzyme extractions, frozen leaf tissue (200 mg) was ground to a fine powder in liquid nitrogen and homogenized in 2 mL of ice-cold extraction buffer (50 mM sodium phosphate buffer, pH 7.0, containing 1 mM EDTA, 1% polyvinylpyrrolidone (PVP), and 1 mM phenylmethylsulfonyl fluoride (PMSF). The homogenate was centrifuged at 12,000 \u0026times; g for 20 min at 4\u0026deg;C, and the supernatant was used as crude enzyme extract for activity assays. Total protein concentration was determined by the Bradford method (Bradford, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1976\u003c/span\u003e) using bovine serum albumin as a standard.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDefense-Related Enzyme Activities\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003ePhenylalanine Ammonia-Lyase (PAL) Assay\u003c/h2\u003e \u003cp\u003ePAL activity was measured following the method of Zucker (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1965\u003c/span\u003e). The reaction mixture contained 0.2 mL enzyme extract, 1 mL of 50 mM Tris-HCl buffer (pH 8.8), and 0.5 mL of 20 mM L-phenylalanine. After incubation at 37\u0026deg;C for 60 min, the reaction was stopped by adding 0.1 mL of 5 M HCl. The absorbance was measured at 290 nm against a blank without L-phenylalanine. PAL activity was expressed as \u0026micro;mol of trans-cinnamic acid produced per hour per mg protein (ε\u0026thinsp;=\u0026thinsp;17,400 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePolyphenol Oxidase (PPO) Assay\u003c/h2\u003e \u003cp\u003ePPO activity was assayed according to Srivastava et al. (1987). The reaction mixture contained 0.1 mL enzyme extract and 2 mL of 50 mM sodium phosphate buffer (pH 6.5) containing 20 mM catechol. The increase in absorbance at 420 nm was monitored over 3 min. PPO activity was defined as the change in absorbance per minute per mg protein (ΔA\u003csub\u003e420\u003c/sub\u003e min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e protein).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePeroxidase (PO) Assay\u003c/h2\u003e \u003cp\u003ePO activity was determined using the guaiacol method (Hammerschmidt et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). The reaction mixture consisted of 0.1 mL enzyme extract, 1.5 mL of 50 mM sodium phosphate buffer (pH 6.5), 0.2 mL of 20 mM guaiacol, and 0.2 mL of 12.3 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The increase in absorbance at 470 nm was recorded over 3 min. PO activity was expressed as ΔA₄₇₀ min⁻\u0026sup1; mg⁻\u0026sup1; protein (ε\u0026thinsp;=\u0026thinsp;26.6 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSuperoxide Dismutase (SOD) Assay\u003c/h2\u003e \u003cp\u003eSOD activity was measured by the nitroblue tetrazolium (NBT) photochemical method (Beauchamp and Fridovich, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1971\u003c/span\u003e). The reaction mixture (3 mL) contained 50 mM sodium phosphate buffer (pH 7.8), 13 mM methionine, 75 \u0026micro;M NBT, 2 \u0026micro;M riboflavin, 0.1 mM EDTA, and 50 \u0026micro;L enzyme extract. The reaction was initiated by illuminating the tubes with a fluorescent lamp for 15 min, after which absorbance was measured at 560 nm. One unit of SOD activity was defined as the amount of enzyme required to inhibit NBT photoreduction by 50%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eCatalase (CAT) Assay\u003c/h2\u003e \u003cp\u003eCAT activity was determined by monitoring the decomposition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Aebi, 1984). The reaction mixture contained 50 mM sodium phosphate buffer (pH 7.0), 10 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and 50 \u0026micro;L enzyme extract. The decrease in absorbance at 240 nm was recorded over 3 min. CAT activity was expressed as \u0026micro;mol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposed per minute per mg protein (ε\u0026thinsp;=\u0026thinsp;39.4 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of Defense-Related Metabolites\u003c/h2\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003eTotal Phenolic Content\u003c/h2\u003e \u003cp\u003eTotal phenolics were extracted from 100 mg of freeze-dried leaf powder with 2 mL of 80% methanol at 4\u0026deg;C for 2 h. After centrifugation (10,000 \u0026times; g, 15 min), the supernatant was collected. Phenolic content was determined using the Folin\u0026ndash;Ciocalteu reagent (Singleton and Rossi, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1965\u003c/span\u003e). An aliquot (0.2 mL) of extract was mixed with 1 mL of 10% Folin\u0026ndash;Ciocalteu reagent and 0.8 mL of 7.5% sodium carbonate. After incubation at room temperature for 30 min, absorbance was measured at 765 nm. Results were expressed as mg gallic acid equivalents (GAE) per g dry weight.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eFlavonoid Content\u003c/h2\u003e \u003cp\u003eFlavonoids were quantified by the aluminum chloride colorimetric method (Zhishen et al., 1999). Methanolic extract (0.5 mL) was mixed with 0.1 mL of 10% AlCl\u003csub\u003e3\u003c/sub\u003e, 0.1 mL of 1 M potassium acetate, and 2.8 mL of distilled water. After incubation at room temperature for 30 min, absorbance was measured at 415 nm. Results were expressed as mg quercetin equivalents (QE) per g dry weight.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eLignin Content\u003c/h2\u003e \u003cp\u003eLignin was quantified using the acetyl bromide method (Fukushima and Hatfield, 2001). Cell wall residues were prepared by washing leaf tissue sequentially with phosphate buffer, methanol, and acetone. Dried cell wall material (10 mg) was digested in 1 mL of 25% acetyl bromide in glacial acetic acid at 70\u0026deg;C for 30 min. After cooling, the mixture was diluted with 2 mL of acetic acid and 1 mL of 2 M NaOH, and then made up to 10 mL with acetic acid. Absorbance was measured at 280 nm, and lignin content was calculated using a molar extinction coefficient of 17.2 g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e L cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eQuantification of Oxidative Stress Markers and Antioxidants\u003c/h2\u003e \u003cdiv id=\"Sec26\" class=\"Section4\"\u003e \u003ch2\u003eHydrogen Peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) Content\u003c/h2\u003e \u003cp\u003eH₂O₂ was quantified according to Velikova et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Leaf tissue (100 mg) was homogenized in 2 mL of 0.1% trichloroacetic acid (TCA). After centrifugation (12,000 \u0026times; g, 15 min), 0.5 mL of supernatant was mixed with 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) and 1 mL of 1 M KI. Absorbance was measured at 390 nm, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration was determined from a standard curve.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eLipid Peroxidation (Malondialdehyde Content)\u003c/h2\u003e \u003cp\u003eLipid peroxidation was estimated by measuring malondialdehyde (MDA) content using the thiobarbituric acid (TBA) method (Heath and Packer, 1968). Leaf tissue (100 mg) was homogenized in 2 mL of 0.1% TCA. After centrifugation, 1 mL of supernatant was mixed with 2 mL of 0.5% TBA in 20% TCA. The mixture was heated at 95\u0026deg;C for 30 min, cooled on ice, and centrifuged. Absorbance was measured at 532 nm and corrected for non-specific absorbance at 600 nm. MDA content was calculated using an extinction coefficient of 155 mM\u003csup\u003e\u0026minus;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eProline Content\u003c/h2\u003e \u003cp\u003eProline was determined by the acid-ninhydrin method (Bates et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1973\u003c/span\u003e). Leaf tissue (100 mg) was homogenized in 2 mL of 3% sulfosalicylic acid and centrifuged. The supernatant (0.5 mL) was reacted with 0.5 mL of glacial acetic acid and 0.5 mL of acid-ninhydrin reagent at 100\u0026deg;C for 1 h. After cooling, the mixture was extracted with 1 mL of toluene, and absorbance of the toluene phase was measured at 520 nm. Proline concentration was calculated from a standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eReduced Glutathione (GSH) Content\u003c/h2\u003e \u003cp\u003eGSH was quantified according to Griffith (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). Leaf tissue (100 mg) was homogenized in 2 mL of 5% sulfosalicylic acid and centrifuged. The supernatant (0.5 mL) was mixed with 0.5 mL of 0.1 M phosphate buffer (pH 7.5) containing 5 mM EDTA, 0.2 mL of 6 mM 5,5\u0026prime;-dithiobis-(2-nitrobenzoic acid) (DTNB), and 0.2 mL of NADPH (0.4 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The change in absorbance at 412 nm was monitored, and GSH content was expressed as nmol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e fresh weight.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhytohormone Extraction and Quantification\u003c/h3\u003e\n\u003cp\u003eJasmonic acid (JA) was extracted from frozen leaf tissue (500 mg) according to Zhang et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) with minor modifications. Samples were ground in liquid nitrogen and homogenized in 5 mL ice-cold 80% methanol containing 1% polyvinylpyrrolidone (PVP) and 10 mg/L butylated hydroxytoluene (BHT). After overnight stirring at 4\u0026deg;C in darkness, homogenates were centrifuged (10,000 \u0026times; g, 20 min, 4\u0026deg;C). The pellet was re-extracted with 3 mL cold 80% methanol for 2 h. Combined supernatants were evaporated to dryness (35\u0026deg;C, rotary evaporator). The residue was resuspended in 2 mL 0.1 M phosphate buffer (pH 8.0) and partitioned twice against ethyl acetate. The aqueous phase was adjusted to pH 2.5 with 1 M HCl and extracted three times with 2 mL diethyl ether. Pooled ether phases were evaporated under nitrogen. For quantification, the dried extract was dissolved in 1 mL methanol, mixed with 2 mL reagent (5% zinc acetate, 5% potassium ferrocyanide), incubated (30\u0026deg;C, 30 min), and absorbance read at 508 nm. JA concentration was calculated from a standard curve (0-100 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Sigma-Aldrich). Recovery averaged 82.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1% .\u003c/p\u003e \u003cp\u003e \u003cb\u003eSalicylic acid (SA)\u003c/b\u003e was extracted following Warrier et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Frozen leaf tissue (300 mg) was homogenized in 3 mL 90% methanol containing 0.5% sodium metabisulfite, sonicated (15 min, 4\u0026deg;C), and centrifuged (12,000 \u0026times; g, 15 min). The pellet was re-extracted with 2 mL 90% methanol. Combined supernatants were evaporated to dryness (40\u0026deg;C, vacuum). The residue was dissolved in 2 mL 5% trichloroacetic acid (TCA) and centrifuged (10,000 \u0026times; g, 10 min). The supernatant was partitioned twice against 2 mL ethyl acetate:cyclopentane (1:1). Organic phases were evaporated under nitrogen. The dried extract was dissolved in 1 mL ethanol, mixed with 2 mL 0.1% ferric chloride in 50% ethanol, incubated (10 min, room temperature), and absorbance measured at 540 nm. SA concentration was determined using a standard curve (0\u0026ndash;50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Sigma-Aldrich). Recovery averaged 88.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8% .\u003c/p\u003e \u003cp\u003eEthylene production was quantified colorimetrically following the method of Larue and Kurz (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1973\u003c/span\u003e) with modifications for potato leaf tissue. Leaf discs (100 mg fresh weight) were incubated in 10 mL gas-tight vials at 25\u0026deg;C for 2 h to allow ethylene accumulation. A 1 mL headspace sample was injected into 1 mL of 0.05 M acidified KMnO\u003csub\u003e4\u003c/sub\u003e to oxidize ethylene to formaldehyde. After 10 min, excess KMnO\u003csub\u003e4\u003c/sub\u003e was reduced with 0.2 mL of 0.1 M NaHSO\u003csub\u003e3\u003c/sub\u003e, and a 0.5 mL aliquot was mixed with 2 mL of chromotropic acid reagent (0.5% in concentrated H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e). The mixture was heated at 100\u0026deg;C for 30 min, cooled, and absorbance measured at 570 nm. Ethylene concentration was determined from a standard curve (0-100 nmol) and expressed as nmol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Recovery averaged 85.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2%, and the detection limit was 2.5 nmol per vial (Larue and Kurz, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1973\u003c/span\u003e; Cristescu et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analyses\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using R (v4.3.2). Data normality and homogeneity of variances were verified with Shapiro\u0026ndash;Wilk and Levene's tests; non-normal data were log-transformed prior to analysis. \u003cem\u003eIn vitro\u003c/em\u003e inhibition and greenhouse disease parameters were analyzed by one-way ANOVA followed by Tukey's HSD (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Root colonization was assessed by two-way ANOVA (strain \u0026times; inoculation mode) with explicit testing of the interaction term to evaluate synergy. Longitudinal greenhouse data were fitted with linear mixed models (treatment fixed, block random), and field disease data were analyzed using generalized linear mixed models with binomial distribution. Biochemical parameters and phytohormones were subjected to two-way ANOVA (treatment \u0026times; time), with Tukey's HSD applied at each time point; phytohormone data were additionally compared to controls using Dunnett's test. Pearson correlations were calculated between biochemical markers and AUDPC. Synergy factors were computed as [BDR \u0026ndash; control] / [(BaC21 \u0026ndash; control) + (DS17R \u0026ndash; control)], with values\u0026thinsp;\u0026gt;\u0026thinsp;1 indicating synergy. Significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, with Benjamini\u0026ndash;Hochberg correction for multiple comparisons. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec33\" class=\"Section2\"\u003e \u003ch2\u003eIdentification and Characterization of Bacterial Strains\u003c/h2\u003e \u003cp\u003eA total of 22 \u003cem\u003eBacillus\u003c/em\u003e isolates were recovered from the potato rhizosphere and identified by partial sequencing of the 16S rRNA gene (Table\u0026nbsp;1). The isolates displayed considerable taxonomic diversity, comprising four distinct species and a group of strains that could not be assigned to a described species. \u003cem\u003eBacillus licheniformis\u003c/em\u003e was the most prevalent species, represented by five isolates (BaC1, BaC5, BaC9, BaC12, BaC15). \u003cem\u003eBacillus subtilis\u003c/em\u003e and \u003cem\u003eBacillus megaterium\u003c/em\u003e were each represented by three isolates (BaC6, BaC10, BaC13 and BaC2, BaC11, BaC14, respectively). Three isolates (BaC8, BaC16, BaC21) were identified as \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e, a species well-known for its biocontrol and plant growth-promoting properties. The remaining eight isolates (BaC3, BaC4, BaC7, BaC17, BaC18, BaC19, BaC20, BaC22) could not be unequivocally assigned to a described species based on the 16S rRNA gene alone and were therefore designated as \u003cem\u003eBacillus\u003c/em\u003e sp., reflecting either the presence of underrepresented taxa or the inherent limitations of single-gene resolution within the \u003cem\u003eBacillus\u003c/em\u003e complex. This taxonomic richness provided a diverse panel for subsequent functional screening, from which strain BaC21 (\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e) emerged as the lead candidate for consortium development owing to its superior antagonistic and plant-growth-promoting traits.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTaxonomic distribution of \u003cem\u003eBacillus\u003c/em\u003e isolates from potato rhizosphere based on 16S rRNA gene sequencing.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIdentification (16S rRNA)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIsolates\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBaC8, BaC16, BaC21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. licheniformis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBaC1, BaC5, BaC9, BaC12, BaC15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. megaterium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBaC2, BaC11, BaC14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. subtilis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBaC6, BaC10, BaC13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacillus\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBaC3, BaC4, BaC7, BaC17, BaC18, BaC19, BaC20, BaC22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCorrespondence Analysis of\u003c/b\u003e \u003cb\u003eBacillus\u003c/b\u003e \u003cb\u003eisolates\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHierarchical clustering of the \u003cem\u003eBacillus\u003c/em\u003e strains (BaC1-BaC22) revealed distinct groups that correlate with their ecological origin and antagonistic traits, thereby illustrating a clear niche-functional relationship (Fig.\u0026nbsp;1). Cluster 1 (BaC18 group), predominantly composed of rhizosphere strains, is characterized by a functional profile geared toward root nutrient mobilization and direct pathogen suppression, as evidenced by elevated cellulase and chitinase activities in strains such as BaC4 and BaC18, coupled with moderate IAA production (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Conversely, Cluster 2 (BaC17 group), which includes phyllosphere and rhizoplane strains, employs a more aggressive chemical arsenal; it is defined by strong volatile-mediated inhibition, high lipase and protease activities, and significant HCN production, suggesting a specialized strategy for targeting both foliar and soil-borne pathogens. Cluster 3 (BaC19 group) further underscores the potency of phyllosphere and rhizoplane isolates, with BaC21 emerging as a standout strain due to its dual antagonistic activity via both diffusible and volatile compounds, supported by high production of the phytohormones SA and IAA. Meanwhile, Cluster 4 (BaC22 group) largely reinforces the enzymatic, non-volatile strategy observed in Cluster 1, highlighting the consistency of this functional guild within the rhizosphere. Collectively, a synergistic division of labor is evident: whereas rhizosphere-associated clusters (1 and 4) primarily rely on enzymatic disruption of pathogen cell walls, the phyllosphere/rhizoplane clusters (2 and 3) prioritize volatile suppression and phytochemical signaling. Ultimately, the identification of four strains notably BaC21 that exhibit potent dual antagonism through both diffusible and volatile mechanisms highlights them as particularly promising biocontrol agents capable of deploying a multifaceted defensive strategy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn Vitro\u003c/b\u003e \u003cb\u003eAntagonism Against\u003c/b\u003e \u003cb\u003ePhytophthora infestans\u003c/b\u003e\u003c/p\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eInhibition by Diffusible and Volatile Compounds\u003c/h2\u003e \u003cp\u003eA total of 22 \u003cem\u003eBacillus\u003c/em\u003e isolates recovered from potato rhizosphere were screened for their ability to inhibit \u003cem\u003ePhytophthora infestans\u003c/em\u003e through three complementary mechanisms: direct confrontation in dual culture (inhibition zone, mm), production of diffusible antimicrobial metabolites (cell-free filtrate, % inhibition), and emission of volatile organic compounds (VOCs, % inhibition) (Table\u0026nbsp;2). Inhibition zones ranged from 8.00 mm (BaC19) to 18.80 mm (BaC21). Isolate BaC21 exhibited the largest inhibition zone (18.80 mm) and was placed in the highest statistical group, significantly outperforming all other strains. A second tier of highly effective isolates included BaC13 (16.00 mm), BaC15 (16.20 mm), and BaC18 (15.66 mm), all of which exceeded 15 mm. Moderately active strains such as BaC6 (14.65 mm), BaC4 (13.76 mm), and BaC8 (13.28 mm) formed overlapping statistical groups. The least effective isolates, including BaC7 (8.36 mm), BaC19 (8.00 mm), and BaC20 (8.10 mm), produced inhibition zones below 10 mm and were statistically similar to one another but significantly lower than the top performers. Inhibition by VOCs ranged from 12.50% (BaC5) to 91.66% (BaC21). Again, BaC21 ranked highest (91.66%), demonstrating exceptional volatile antagonism. BaC15 (81.00%) and BaC18 (78.75%) formed the next tier, followed by a cluster of isolates including BaC13 (71.66%), BaC6 (71.42%), and BaC20 (74.16%). Isolates with moderate volatile activity (50\u0026ndash;65% inhibition) included BaC16 (64.91%), BaC17 (61.08%), and BaC1 (53.76%). The lowest volatile producers were BaC2 (42.85%), BaC5 (12.50%), and BaC7 (15.00%), with BaC5 and BaC7 statistically indistinguishable and significantly inferior to all others. Inhibition by secreted metabolites ranged from 28.5% (BaC5) to 76.8% (BaC21). BaC21 again occupied the exclusive top position (76.8%). The second tier comprised BaC15 (68.5%) and BaC18 (65.2%). A third group included BaC13 (62. %), BaC6 (58. %), BaC14 (58.1%), BaC20 (53. %), BaC16 (52.3%), and BaC17 (50.8%). Moderately active isolates such as BaC8 (48.5%), BaC11 (42.8%), and BaC19 (42.5%) showed inhibition between 40% and 50%. The least effective strains included BaC1 (42.3%), BaC2 (38.5%), BaC3 (36.2%), BaC4 (35.8%), BaC22 (34.5%), BaC7 (30.2%), and BaC5 (28.5%), with BaC5 and BaC7 forming the lowest statistical group. Across all three assays, a clear hierarchy emerged. Isolate BaC21 consistently exhibited the highest inhibition in dual culture (18.80 mm), volatile assay (91.66%), and cell-free filtrate assay (76.8%), placing it in the exclusive top statistical group for all three parameters. This multifaceted antagonistic capacity indicates that BaC21 produces a diverse arsenal of diffusible and volatile antimicrobial compounds effective against \u003cem\u003eP. infestans\u003c/em\u003e. Several other isolates, notably BaC15, BaC18, BaC13, and BaC6, also demonstrated strong activity, particularly in volatile and filtrate assays, but none matched the consistency and magnitude of BaC21. Strains such as BaC5, BaC7, and BaC22 were consistently among the least effective across all assays.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e antagonistic activity of \u003cem\u003eBacillus\u003c/em\u003e spp. isolates against \u003cem\u003ePhytophthora infestans\u003c/em\u003e through dual culture, cell free filtrate and volatile compounds\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBacterial strains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eInhibition of \u003cem\u003eP. infestans\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDual culture (mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003evolatile substances (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCell free filtrate (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.00\u003csup\u003eefg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.79\u003csup\u003ecde\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003csup\u003efg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.50\u0026thinsp;\u0026plusmn;\u0026thinsp;2.20\u003csup\u003edef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40.94\u0026thinsp;\u0026plusmn;\u0026thinsp;4.83\u003csup\u003egh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e36.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.76\u0026thinsp;\u0026plusmn;\u0026thinsp;3.46\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.26\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83\u003csup\u003edefg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.50\u0026thinsp;\u0026plusmn;\u0026thinsp;3.11\u003csup\u003ek\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e71.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.00\u0026thinsp;\u0026plusmn;\u0026thinsp;4.31\u003csup\u003ejk\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e51.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003csup\u003eefg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37.75\u0026thinsp;\u0026plusmn;\u0026thinsp;2.44\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003csup\u003eefg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003csup\u003efg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003csup\u003efg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00\u003csup\u003ecde\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003csup\u003egh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.20\u0026thinsp;\u0026plusmn;\u0026thinsp;2.84\u003csup\u003edefg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.29\u0026thinsp;\u0026plusmn;\u0026thinsp;2.15\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e39.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003csup\u003eefg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e71.66\u0026thinsp;\u0026plusmn;\u0026thinsp;3.47\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.20\u0026thinsp;\u0026plusmn;\u0026thinsp;3.66\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e81.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e68.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003csup\u003ecde\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2.13\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003csup\u003ecde\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.08\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e78.75\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e65.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.90 \u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50.91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.10\u0026thinsp;\u0026plusmn;\u0026thinsp;2.60\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e74.16\u0026thinsp;\u0026plusmn;\u0026thinsp;6.15\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e53.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e91.66\u0026thinsp;\u0026plusmn;\u0026thinsp;3.82\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e76.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003efg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003csup\u003eij\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eData with different letters in the same column are significantly different after ANOVA at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 using Turkey\u0026rsquo;s HSD test.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eCompatibility and Co-culture Dynamics\u003c/h3\u003e\n\u003cp\u003eThe compatibility between \u003cem\u003eBacillus\u003c/em\u003e sp. BaC21 and \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e DS17R was evaluated through cross-streak assays and co-culture dynamics (Supplementary Table S2). On nutrient agar, the two strains grew contiguously without any visible inhibition zone at their intersection, indicating the absence of direct antagonism. In liquid medium, co-culture growth kinetics closely followed those of the faster-growing partner (DS17R), reaching stationary phase densities comparable to the individual cultures (final OD\u003csub\u003e600\u003c/sub\u003e values: BaC21 alone 1.85; DS17R alone 1.92; co-culture 1.89). The final pH of the co-culture (6.8) was intermediate between that of BaC21 (7.1) and DS17R (6.5), suggesting no metabolic incompatibility or production of inhibitory by-products. These results demonstrate that BaC21 and DS17R are fully compatible for co-application, a prerequisite for the rational design of a stable functional consortium.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGrowth dynamics of\u003c/b\u003e \u003cb\u003eBacillus\u003c/b\u003e \u003cb\u003esp. BaC21 and\u003c/b\u003e \u003cb\u003ePseudomonas fluorescens\u003c/b\u003e \u003cb\u003eDS17R in mono- and co-culture\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe growth dynamics of \u003cem\u003eBacillus\u003c/em\u003e sp. BaC21 and \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e DS17R were monitored over 96 hours in both mono- and co-culture to assess their compatibility and potential ecological interactions (Fig.\u0026nbsp;2). Both strains exhibited typical sigmoidal growth curves, reaching stationary phase by 48\u0026ndash;72 hours. For \u003cem\u003eBacillus\u003c/em\u003e BaC21, population densities in mono-culture increased from 6.02 log\u003csub\u003e10\u003c/sub\u003e CFU/mL at inoculation to 8.55 log\u003csub\u003e10\u003c/sub\u003e CFU/mL at 96 h. When co-cultured with \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e growth was nearly identical, with final densities of 8.48 log\u003csub\u003e10\u003c/sub\u003e CFU/mL. Similarly, \u003cem\u003ePseudomonas\u003c/em\u003e DS17R reached 8.21 log\u003csub\u003e10\u003c/sub\u003e CFU/mL in mono-culture and 8.31 log\u003csub\u003e10\u003c/sub\u003e CFU/mL in co-culture at 96 h. Statistical comparisons at each time point revealed no significant differences between mono- and co-culture conditions for either strain (unpaired t-tests, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05 at all time points; asterisks denote non-significance, \"ns\"). The absence of growth inhibition or enhancement indicates neutral coexistence without direct antagonism or strong metabolic interference. Furthermore, cross-streak assays confirmed the absence of any inhibition zone (Supplementary Table S2), corroborating the liquid culture results. These findings demonstrate that BaC21 and DS17R are fully compatible for co-application, as neither strain suppresses the other's proliferation. This ecological compatibility is a prerequisite for the rational design of a stable, functional consortium and supports the hypothesis that these two strains can occupy complementary niches without competing aggressively for resources.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eEffects of strain identity and consortium formation on root colonization dynamics\u003c/h3\u003e\n\u003cp\u003eThe figure illustrates the temporal dynamics of rhizosphere colonization by \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e, both individually and in a consortium, across three post-inoculation time points (Day 3, 7, and 14). At Day 3, \u003cem\u003eBacillus\u003c/em\u003e alone achieved an average colonization of 5.82 log\u003csub\u003e10\u003c/sub\u003e CFU/g, which was slightly lower than \u003cem\u003eBacillus\u003c/em\u003e in the consortium (6.24 log\u003csub\u003e10\u003c/sub\u003e CFU/g). The Tukey post-hoc test indicates that these two treatments are not significantly different. \u003cem\u003ePseudomonas\u003c/em\u003e alone showed 6.58, while \u003cem\u003ePseudomonas\u003c/em\u003e in consortium reached 6.91; these two treatments also did not differ significantly. Overall, at this early stage, consortium inoculation trends slightly higher but differences are not statistically significant. By Day 7, colonization increased for all groups. \u003cem\u003eBacillus\u003c/em\u003e alone reached 6.95, whereas \u003cem\u003eBacillus\u003c/em\u003e in consortium reached 7.8, indicating a significant enhancement of colonization when Bacillus is co-inoculated. \u003cem\u003ePseudomonas\u003c/em\u003e alone measured 7.42 and in consortium 8.15, showing a similar trend. The test used onfirm that consortium treatments (both \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e) are significantly higher than the respective single-strain inoculations. The interaction between strain and consortium at Day 7 is highlighted by the p-value of 0.019, suggesting a synergistic effect in consortium formation. At Day 14, the highest colonization was observed for \u003cem\u003ePseudomonas\u003c/em\u003e in consortium (9.03), followed closely by \u003cem\u003eBacillus\u003c/em\u003e in consortium (8.92). Single inoculations showed slightly lower levels (\u003cem\u003eBacillus\u003c/em\u003e alone: 7.48; \u003cem\u003ePseudomonas\u003c/em\u003e alone: 8.15).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003ch2\u003eGreenhouse Disease Suppression and Plant Growth Promotion\u003c/h2\u003e \u003cp\u003eDisease severity was assessed at 7 and 14 days post-inoculation (dpi), and the area under the disease progress curve (AUDPC) was calculated for each treatment (Fig.\u0026nbsp;4). At 7 dpi, the untreated control showed a mean disease severity of 3.2 (scale 0\u0026ndash;5), which increased to 4.5 by 14 dpi. Single-strain treatments significantly reduced disease: BaC21 lowered severity to 1.5 at 7 dpi and 2.1 at 14 dpi, while DS17R gave values of 1.9 and 2.8, respectively. The BDR consortium was markedly more effective, with severity scores of only 0.6 at 7 dpi and 0.9 at 14 dpi, statistically equivalent to the chemical control Ridomil (0.5 and 0.8). AUDPC values mirrored these trends: control (54.8), BaC21 (25.2), DS17R (32.9), BDR (10.5), and Ridomil (9.8). Different letters in the figure indicate that BDR and Ridomil formed a distinct, highly protected group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the single strains were intermediate and the control was most susceptible. These data confirm that the consortium provides superior, near-chemical protection against late blight under greenhouse conditions.\u003c/p\u003e \u003cp\u003eAll bacterial treatments significantly enhanced potato growth and yield compared to the untreated control, but with distinct patterns reflecting functional complementarity (Table\u0026nbsp;3). Plant height increased from 24.6 cm in the control to 27.6 cm (BaC21), 28.1 cm (BDR), and 28.4 cm (DS17R); all three bacterial treatments were statistically similar and significantly taller than the control. Stem diameter followed a different order: DS17R (13.2 mm) induced the thickest stems, significantly exceeding BDR (12.3 mm) and BaC21 (11.4 mm), while the control remained smallest (9.3). The most striking differences were observed in root proliferation: DS17R alone produced the highest number of main roots (27.3), followed by the BDR consortium (22.2), BaC21 (15.3), and control (13.1). Despite DS17R's superior root stimulation, the consortium yielded the highest tuber production (281.3g per plant), significantly outperforming BaC21 (264.5g) and DS17R (270.1g). This pattern illustrates the synergistic integration of DS17R's root-enhancing capability with BaC21's yield-promoting traits, resulting in a net productivity gain that neither strain achieves alone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGrowth promotion effects of bacteria treatments on potato plants under greenhouse conditions.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlant height (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStem diameter (mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNumber of main roots\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYield per plant (g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e246.3\u0026thinsp;\u0026plusmn;\u0026thinsp;10.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.35\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e264.5\u0026thinsp;\u0026plusmn;\u0026thinsp;12.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDS17R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e270.1\u0026thinsp;\u0026plusmn;\u0026thinsp;11.5\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBDR consortium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.38\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e281.3\u0026thinsp;\u0026plusmn;\u0026thinsp;13.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;6). Different letters within a column indicate significant differences according to Tukey's HSD test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e \u003ch2\u003eField Performance and and Tuber Quality\u003c/h2\u003e \u003cp\u003eField trial data confirmed the superior performance of the BDR consortium under natural infection pressure (Table\u0026nbsp;4). At the first assessment (30 days after planting), all bacterial treatments already reduced disease severity compared to the untreated control (1.2): BaC21 (0.4), DS17R (0.5), BDR (0.2), and Ridomil (0.1). By 60 days, disease had progressed substantially in control plots (3.5) and, to a lesser extent, in single-strain treatments (BaC21 1.8, DS17R 2.1). In contrast, BDR (0.7) and Ridomil (0.5) maintained very low severity, statistically equivalent to each other and significantly lower than all other treatments. At the final assessment (90 days), control plants were nearly destroyed (4.8), while BDR (1.1) and Ridomil (0.9) remained highly protected. The final AUDPC values quantitatively summarize this pattern: control 285, BaC21 142, DS17R 168, BDR 58, and Ridomil 42. The consortium achieved a disease reduction of 79.6% relative to the control, statistically indistinguishable from the chemical fungicide. These results demonstrate the consortium's ability to provide durable, field-relevant protection comparable to conventional fungicides.\u003c/p\u003e \u003cp\u003eConsistent with disease suppression, tuber yield and quality were highest in plots treated with the BDR consortium and Ridomil. Total yield reached 24.8 t.ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for BDR and 26.1 t ha⁻\u0026sup1; for Ridomil a three-fold increase over the untreated control (8.2 t.ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and significantly greater than the single strains (BaC21 18.5, DS17R 15.2). Marketable tuber percentage followed the same hierarchy: BDR (85.3%) and Ridomil (91.2%) were statistically equivalent and both superior to BaC21 (68.2%) and DS17R (61.5%), while the control produced only 42.3% marketable tubers. The consortium's yield advantage over single strains (34% higher than BaC21, 63% higher than DS17R) clearly demonstrates a synergistic effect that translates directly into economic benefit.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDisease progression and yield in field trials\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDisease severity (0\u0026ndash;5 scale)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFinal AUDPC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYield (t ha⁻\u0026sup1;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMarketable tubers (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30 DAP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60 DAP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90 DAP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e285\u0026thinsp;\u0026plusmn;\u0026thinsp;12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e42.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaC21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e142\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e68.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDS17R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e168\u0026thinsp;\u0026plusmn;\u0026thinsp;9\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e61.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBDR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e58\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e85.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRidomil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e42\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e91.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE (n\u0026thinsp;=\u0026thinsp;4 plots per treatment, 36 plants per plot). Different superscript letters within columns indicate significant differences (Tukey's HSD, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003cem\u003eDAP\u0026thinsp;=\u0026thinsp;Days After Planting. AUDPC\u0026thinsp;=\u0026thinsp;Area Under Disease Progress Curve.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec39\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical Defense Responses\u003c/h2\u003e \u003cdiv id=\"Sec40\" class=\"Section3\"\u003e \u003ch2\u003eActivities of Defense-Related Enzymes\u003c/h2\u003e \u003cp\u003eThe activities of five key defense-related enzymes were quantified at 24, 48, and 72 h post-inoculation (hpi) to assess the biochemical basis of resistance (Fig.\u0026nbsp;5). In all cases, the BDR consortium induced the highest enzyme activities, with significant differences denoted by letters (Tukey's HSD, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). For phenylalanine ammonia-lyase (PAL), the gateway enzyme of phenylpropanoid metabolism, BDR-treated plants reached 2.98\u0026micro;mol h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e protein at 48 h \u0026ndash; 6.6-fold higher than the uninfected control (T\u0026ndash;, 0.45) and 2.3-fold higher than the infected control (T+, 1.32). The single strains also elevated PAL activity (BaC21 2.35, DS17R 1.82), but remained significantly below the consortium. Polyphenol oxidase (PPO) and peroxidase (PO) followed similar temporal patterns, peaking at 48 h with BDR values of 1.98 and 1.85 ΔA min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e protein, respectively, exceeding both single strains and controls. Superoxide dismutase (SOD) and catalase (CAT) activities, reflecting antioxidant capacity, were also maximally induced by BDR (SOD 42.8 U.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, CAT 32.5 \u0026micro;mol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 48 h). Across all enzymes, the consortium consistently formed the top statistical group, while the single strains occupied intermediate positions and the infected control (T+) ranked lowest among challenged plants. These data demonstrate that the consortium primes a stronger and more coordinated enzymatic defense response than either strain alone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eAccumulation of Defense-Related Metabolites\u003c/h3\u003e\n\u003cp\u003eThe accumulation of defense-related secondary metabolites mirrored the enzyme activity patterns (Fig.\u0026nbsp;6). Total phenolic content in BDR-treated leaves reached 21.5 mg GAE g⁻\u0026sup1; DW at 48 h \u0026ndash; 4.8-fold higher than T\u0026ndash; (4.5) and 2.3-fold higher than T+ (9.5). BaC21 and DS17R induced intermediate levels (16.8 and 12.5, respectively), with all treatments significantly different from each other. Flavonoid content followed the same hierarchy: BDR (9.8 mg QE g\u003csup\u003e\u0026minus;1\u003c/sup\u003eDW) \u0026gt; BaC21 (7.5) \u0026gt; DS17R (5.5)\u0026thinsp;\u0026gt;\u0026thinsp;T+ (4.2)\u0026thinsp;\u0026gt;\u0026thinsp;T\u0026ndash; (1.9). Lignin deposition, a key structural defense, was also highest in BDR plants (38.5mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW at 48 h), significantly exceeding BaC21 (32.5), DS17R (26.8), and controls. The temporal profiles show maximal accumulation at 48 h for all metabolites, coinciding with the peak of enzyme activities. The consortium's ability to elevate both phenolic and lignin barriers provides a robust chemical and physical defense against pathogen invasion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eOxidative Stress Markers and Antioxidants\u003c/h3\u003e\n\u003cp\u003eOxidative stress markers were assessed to evaluate cellular damage and antioxidant status (Fig.\u0026nbsp;7). Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) levels, indicative of oxidative burst, were highest in the infected control (T+) at 48 h (18.5 \u0026micro;mol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW). Single-strain treatments reduced H₂O₂ accumulation (BaC21 10.5, DS17R 14.5), but the consortium was most effective, lowering H₂O₂ to 8.5 \u0026micro;mol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW a level only 54% higher than the uninfected control (T\u0026ndash;, 5.5). Malondialdehyde (MDA), a product of lipid peroxidation, followed the same trend: T+ (25.5 nmol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW) \u0026gt; DS17R (20.5) \u0026gt; BaC21 (15.5)\u0026thinsp;\u0026gt;\u0026thinsp;BDR (12.5)\u0026thinsp;\u0026gt;\u0026thinsp;T\u0026ndash; (8.2). Conversely, the osmoprotectant proline and the antioxidant glutathione (GSH) were most abundant in BDR-treated plants. Proline peaked at 48 h in BDR (3.25 \u0026micro;mol.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW), significantly exceeding BaC21 (2.65), DS17R (2.15), and T+ (1.85). GSH content was also highest in BDR (265 nmol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW at 48 h), representing a 3.0-fold increase over T+ (88) and a 1.6-fold increase over the best single strain (BaC21 215). These results indicate that the consortium not only limits oxidative damage but also boosts the plant's antioxidant capacity, contributing to cellular homeostasis under pathogen attack.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003ePhotosynthetic Pigment and Cysteine Content\u003c/h3\u003e\n\u003cp\u003ePhysiological fitness was assessed by measuring chlorophyll a content (14 dpi) and cysteine (reduced glutathione) levels at 24, 48, and 72 h (Fig.\u0026nbsp;8). Chlorophyll a was severely reduced in the infected control (1.52 mg.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW) compared to the uninfected control (2.45). Single-strain treatments partially preserved chlorophyll (BaC21 1.98, DS17R 2.12), but only the BDR consortium maintained chlorophyll levels (2.35) statistically indistinguishable from T\u0026ndash;, indicating that the strong defense activation did not compromise photosynthetic capacity. Cysteine content, a key component of the glutathione antioxidant system, was highest in BDR-treated plants at all time points. At 48 h, BDR reached 265 g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW, significantly above BaC21 (215), DS17R (165), T+ (135), and T\u0026ndash; (88). The temporal increase and sustained elevation of cysteine in consortium-treated plants reflect enhanced redox buffering capacity. Collectively, these data demonstrate that the BDR consortium achieves robust disease resistance while maintaining photosynthetic efficiency and boosting antioxidant potential a physiological optimization that avoids the typical growth-defense trade-off.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDynamics of jasmonic acid (JA), ethylene, and salicylic acid (SA) in potato leaves following bacterial treatments against\u003c/b\u003e \u003cb\u003eP. infestans\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe phytohormone profiling revealed distinct temporal patterns that corroborate the proposed model of sequential defense priming orchestrated by the BDR consortium. Jasmonic acid (JA) levels remained near baseline (approximately 10 ng.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW) in all treatments at 0 h post-inoculation (hpi) (Fig.\u0026nbsp;9). By 6 hpi, however, marked differences emerged: the infected control (T+) reached 35, while single strains Bac and Pse induced 45 and 30 ng.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW, respectively. The BDR consortium elicited the highest JA accumulation at this early time point (60 ng.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW), significantly exceeding all other treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). JA peaked at 12 hpi across all challenged treatments, with BDR again showing the maximum induction (95 ng/g FW), followed by Bac (75), T+ (60), and Pse (50). Thereafter, JA levels gradually declined through 72 hpi, although BDR maintained significantly higher concentrations than other treatments at all time points. Ethylene production followed a remarkably similar trajectory, with BDR inducing the highest levels at 6 hpi (3.8 nL.g\u003csup\u003e\u0026minus;1\u003c/sup\u003eFW) and 12 hpi (6.0 nL.g\u003csup\u003e\u0026minus;1\u003c/sup\u003eFW), significantly outperforming both single strains and the infected control. In contrast, salicylic acid (SA) exhibited a delayed accumulation pattern. SA concentrations remained relatively low in all treatments until 12 hpi, when BDR began to separate from the others (200 ng.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW). The peak of SA accumulation occurred at 48 hpi, where BDR induced 550 ng/g FW significantly higher than Bac (380 ng.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW), T+ (300 ng.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW), Pse (240 ng.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW), and the uninfected control T- (58 ng.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW). This sequential activation early JA/ethylene followed by later SA was unique to the BDR consortium and contrasted with the single strains, which showed less pronounced and less coordinated hormone induction. The statistical letters at each time point confirm that BDR consistently formed the top tier for both early and late hormone responses, while T- remained lowest throughout. These hormonal dynamics provide direct evidence that the consortium primes a temporally integrated defense program, engaging the JA/ethylene pathway during the initial recognition phase and subsequently mobilizing the SA pathway for sustained resistance, thereby avoiding the mutual antagonism often reported between these signaling cascades.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCorrelation Between Biochemical Markers and Disease Resistance\u003c/h3\u003e\n\u003cp\u003ePearson correlation analysis revealed strong relationships between biochemical markers measured at 48 h post-inoculation, phytohormone levels at their respective peak time points, and final disease severity (AUDPC) (Table\u0026nbsp;5). PAL activity showed a highly significant negative correlation with AUDPC (r = -0.89, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), as did PPO (r = -0.85), PO (r = -0.87), SOD (r = -0.82), and CAT (r = -0.79). Total phenolics (r = -0.91), flavonoids (r = -0.88), and lignin (r = -0.84) were also strongly inversely correlated with disease severity. The phytohormone analyses provided complementary insights: JA levels at 12 hpi (the peak of early defense signaling) exhibited a strong negative correlation with AUDPC (r = -0.83, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while ethylene at 12 hpi showed an even stronger correlation (r = -0.87, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). SA levels at 48 hpi, representing the later phase of defense activation, displayed the strongest negative correlation among all hormonal parameters (r = -0.92, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), underscoring the critical role of SA-mediated defenses in determining final disease outcome. Conversely, oxidative stress markers exhibited strong positive correlations with disease severity: H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (r\u0026thinsp;=\u0026thinsp;0.76, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and MDA (r\u0026thinsp;=\u0026thinsp;0.85, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The antioxidants proline (r = -0.81) and GSH (r = -0.86) were negatively correlated with AUDPC. These correlations validate the functional relevance of both the biochemical and hormonal parameters measured: higher defense enzyme activities, secondary metabolite accumulation, and coordinated phytohormone induction are associated with lower disease severity, while elevated oxidative stress markers indicate greater tissue damage. The particularly strong correlation between SA at 48 h and disease reduction (r = -0.92) highlights the importance of sustained SA-mediated defenses in the resistance phenotype conferred by the BDR consortium. The strength and significance of these correlations collectively underscore the mechanistic link between the multifaceted biochemical and hormonal changes induced by the consortium and its protective efficacy.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePearson correlation matrix between biochemical parameters and disease severity (AUDPC)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCorrelation with AUDPC (r)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDefense enzymes (48 h)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAL activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePPO activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePO activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSOD activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCAT activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSecondary metabolites (48 h)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal phenolics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlavonoids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLignin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePhytohormones\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJA (12 h)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEthylene (12 h)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSA (48 h)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOxidative stress markers (48 h)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMDA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAntioxidants (48 h)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGSH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eSynergistic Effects of the BDR Consortium\u003c/h3\u003e\n\u003cp\u003eTo quantify the synergistic interaction between BaC21 and DS17R in the BDR consortium, synergy factors were calculated as the ratio of the consortium's effect to the sum of the individual strains' effects (minus the control) (Table\u0026nbsp;6). A factor\u0026thinsp;\u0026gt;\u0026thinsp;1 indicates synergy. For in vitro inhibition (dual culture), the consortium achieved a synergy factor of 1.08, indicating a modest additive enhancement. Root colonization at 14 days showed a synergy factor of 1.21 for \u003cem\u003eBacillus\u003c/em\u003e and 1.11 for \u003cem\u003ePseudomonas\u003c/em\u003e, demonstrating that co-inoculation improves rhizosphere establishment of both partners. The most pronounced synergy was observed in disease-related parameters: greenhouse disease reduction (1.50), field yield increase (1.62), and marketable tuber percentage (1.58). Biochemical parameters also exhibited synergy: PAL activity at 48 h (1.27), total phenolics (1.28), and GSH content (1.23). The reduction in oxidative damage (MDA) showed a synergy factor of 1.31, indicating that the consortium is more effective at mitigating cellular stress than expected from the sum of individual effects. Notably, phytohormone induction exhibited particularly high synergy factors: JA at 12 h (1.35), ethylene at 12 h (1.38), and SA at 48 h (1.41). These values indicate that the consortium's ability to coordinate early and late defense signaling pathways represents an emergent property that substantially exceeds the additive contributions of the individual strains. The SA synergy factor of 1.41 is especially significant, as it correlates with the strong negative association between SA accumulation and disease severity observed in Table\u0026nbsp;5. These synergy factors collectively demonstrate that the BDR consortium functions as more than the sum of its parts, with emergent properties particularly the coordinated temporal activation of phytohormone signaling\u0026mdash;that enhance biocontrol efficacy, plant growth promotion, and physiological optimization beyond what either strain achieves alone.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSynergistic effects of the BDR consortium compared to single strains\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBaC21 (% of control)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDS17R (% of control)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBDR (% of control)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSynergy factor\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIn vitro antagonism\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDual culture inhibition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e78.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e84.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRoot colonization (day 14)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacillus\u003c/em\u003e\u0026nbsp;population\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e748%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e903%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePseudomonas\u003c/em\u003e\u0026nbsp;population\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e815%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e903%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDisease and yield parameters\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDisease reduction (greenhouse)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e54.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80.8%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYield increase (field)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e125%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e202%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMarketable tubers\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDefense enzymes (48 h)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAL activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e522%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e404%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e662%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSecondary metabolites (48 h)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal phenolics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e373%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e278%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e478%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePhytohormones\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJA (12 h)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e750%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e500%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e950%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEthylene (12 h)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e900%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e600%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1200%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSA (48 h)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e655%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e414%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e948%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAntioxidants and oxidative stress\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGSH content (48 h)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e244%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e188%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e301%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMDA reduction (48 h)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSynergy factor calculated as: [BDR effect / (BaC21 effect\u0026thinsp;+\u0026thinsp;DS17R effect - control effect)]. Values\u0026thinsp;\u0026gt;\u0026thinsp;1 indicate synergistic interaction\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe BDR consortium, combining \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e BaC21 and \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e DS17R, provided superior protection against \u003cem\u003ePhytophthora infestans\u003c/em\u003e through functional complementarity that manifests at three interconnected levels: ecological facilitation in the rhizosphere, temporally coordinated phytohormone signaling that circumvents SA-JA antagonism, and multilayered biochemical execution through the phenylpropanoid pathway all achieved without the growth-defense trade-off that typically constrains plant immunity. The enhanced root colonization observed upon co-inoculation represents the foundational layer of this synergy. While DS17R alone demonstrated superior rhizosphere competence, its co-application with BaC21 increased \u003cem\u003ePseudomonas\u003c/em\u003e populations by 11% and, more strikingly, boosted \u003cem\u003eBacillus\u003c/em\u003e populations by nearly 20% over its single-strain level. This reciprocal facilitation mirrors recent findings with \u003cem\u003eBacillus-Lysobacter\u003c/em\u003e consortia, where metabolic cross-feeding enhanced biofilm formation and stability (Sun et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The underlying mechanism likely involves pyoverdine siderophores produced by \u003cem\u003ePseudomonas\u003c/em\u003e compounds that not only chelate iron but also act as bacterial determinants of induced systemic resistance (ISR) in multiple plant species (van Loon et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). By facilitating iron acquisition for both strains, these siderophores may simultaneously enhance rhizosphere establishment while priming the plant for defense. This dual function aligns with the concept that successful biocontrol consortia operate not merely as collections of antagonistic strains but as integrated units with emergent ecological properties. The temporal dynamics of phytohormone induction distinguish the consortium fundamentally from single-strain treatments. The early peak of jasmonic acid (JA, 95 ng/g FW at 12 h) and ethylene (6.0 nL/g FW/h at 12 h) in BDR-treated plants, followed by sustained salicylate (SA) accumulation (550 ng/g FW at 48 h), represents a precisely orchestrated immune signature that circumvents the well-documented mutual antagonism between these signaling cascades (Zhang et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Leon-Reyes et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). This pattern is particularly significant given that pathogens and herbivores often manipulate SA-JA crosstalk to suppress plant defenses; the mealybug \u003cem\u003ePhenacoccus solenopsis\u003c/em\u003e, for instance, exploits this antagonism by increasing SA to suppress JA-dependent resistance (Zhang et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The consortium's ability to activate both pathways sequentially rather than antagonistically likely reflects ethylene's modulating role, as ethylene has been shown to render SA-JA antagonism NPR1-independent, effectively overriding the canonical suppression mechanism (Leon-Reyes et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The strong negative correlation between SA at 48 h and AUDPC (r = -0.92) confirms that this delayed SA burst is functionally critical for resistance, consistent with the established role of SA in systemic acquired resistance against biotrophic and hemibiotrophic pathogens. This primed hormonal state translated into potent biochemical execution through the phenylpropanoid pathway. PAL activity in BDR-treated plants reached levels comparable to those induced by methyl jasmonate in date palm cultures, where 200 \u0026micro;M MeJA increased PAL 3.65-fold and enhanced accumulation of catechin, caffeic acid, and p-coumaric acid (Ben Romdhane et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The subsequent elevation of PPO activity is particularly noteworthy, as PPO-generated quinones serve dual functions: direct antimicrobial activity and spatial regulation of programmed cell death to contain pathogen spread (Ben Romdhane et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The lignin accumulation observed aligns with studies on pear fruit showing that defense elicitors activate calcium signaling to upregulate PAL, C4H, 4CL, and CAD, thereby channeling phenylpropanoid flux toward lignin biosynthesis (Guo et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This coordinated activation of multiple pathway enzymes rather than isolated induction of PAL distinguishes the consortium's effect and explains the robust physical barrier formed against pathogen ingress. The preservation of photosynthetic capacity (chlorophyll a maintained at 2.35 mg/g FW, indistinguishable from uninfected controls) alongside elevated antioxidant metabolites (cysteine 3.0-fold over infected controls) addresses a fundamental constraint in plant immunity: the growth-defense trade-off. While methyl jasmonate treatment can reduce cell viability by 35% at high concentrations due to oxidative stress (Ben Romdhane et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), the consortium achieved defense activation without such penalty. This physiological optimization likely reflects the continuous presence of growth-promoting rhizobacteria sustaining primary metabolism, a key advantage over chemical inducers that impose metabolic costs. The synergy factors calculated for disease reduction (1.50) and yield increase (1.62) substantially exceed those reported for many two-strain combinations and align with recent advances in consortium design. A four-microbe consortium recently demonstrated 87% disease control against \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e in cluster bean, with comparable upregulation of PAL (1.93-fold), PPO (2.69-fold), and total phenolics (2.11-fold) (Singh et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The consistency of these biochemical responses across different pathosystems and consortium compositions suggests that enhanced phenylpropanoid metabolism represents a conserved mechanism of consortium-mediated resistance. Similarly, the cooperative interactions observed between \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e in our study echo recent findings with \u003cem\u003eBacillus-Lysobacter\u003c/em\u003e consortia, where spent medium from one strain enhanced antifungal metabolite production in the partner, demonstrating that metabolic cooperation not merely independent complementary actions underlies emergent biocontrol properties (Sun et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Several mechanistic questions warrant further investigation. Whether the observed synergy stems primarily from independent complementary actions or from active molecular dialogue between consortium members could be addressed through metabolomic profiling of co-cultures. Recent evidence suggests that co-cultivation can induce production of metabolites not detected in axenic cultures, raising the possibility that interspecific interactions generate novel bioactive compounds (Sun et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Additionally, the role of calcium signaling as a convergence point for elicitor perception given that bacterial siderophores and LPS trigger rapid Ca\u0026sup2;⁺ fluxes in plant cells (Guo et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; van Loon et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) merits investigation as a potential integrator of the consortium's effects. In the present study, the BDR consortium functions as an integrated ecological unit wherein DS17R establishes rhizosphere competence and initiates ISR signaling while BaC21 amplifies both direct antagonism and biochemical defense execution through temporally coordinated phytohormone activation. By achieving field efficacy comparable to a chemical fungicide while avoiding the growth-defense trade-off, this work provides a mechanistic framework for rationally designing microbial consortia that enhance plant immunity through synergistic interactions across ecological, hormonal, and metabolic levels.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that the rationally designed BDR consortium, comprising \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e BaC21 and \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e DS17R, provides synergistic protection against potato late blight through multilayered mechanisms that operate across ecological, hormonal, and biochemical levels. The consortium's superior efficacy emerges from functional complementarity between the two strains: DS17R establishes rhizosphere competence and initiates ISR signaling, while BaC21 contributes potent direct antibiosis and amplifies defense execution. Co-inoculation enhanced root colonization of both partners, with \u003cem\u003eBacillus\u003c/em\u003e populations increasing nearly 20% a reciprocal facilitation likely mediated by pseudomonad siderophores that improve iron availability and \u003cem\u003eBacillus\u003c/em\u003e lipopeptides that modify root surface properties. The temporally coordinated phytohormone response early JA/ethylene (6\u0026ndash;12 h) followed by sustained SA accumulation (48 h) circumvents the well-documented antagonism between these signaling cascades and enables broad-spectrum defensive readiness. This primed state translates into potent biochemical execution through PAL-driven phenylpropanoid metabolism, PPO-generated quinone toxicity, and lignin deposition that reinforces physical barriers. Critically, this robust defense activation occurs without compromising photosynthetic performance, as BDR-treated plants maintained chlorophyll levels equivalent to uninfected controls while enhancing antioxidant capacity through elevated glutathione precursors. The strong correlations between biochemical parameters and disease reduction validate the functional relevance of these responses. By achieving field efficacy comparable to a chemical fungicide and synergy factors exceeding 1.5 for disease reduction and yield increase, the BDR consortium addresses the inconsistency limitations of single-strain biocontrol agents. This work provides a mechanistic framework for the rational design of microbial consortia in sustainable agriculture, wherein emergent properties arise not merely from additive effects but from genuine synergy across ecological, hormonal, and metabolic levels. Future research should explore the molecular dialogue between consortium members and develop stable formulations for commercial application.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAUDPC: Area Under the Disease Progress Curve\u003c/p\u003e\n\u003cp\u003eBHT: Hydroxytoluene\u003c/p\u003e\n\u003cp\u003eCAT: Catalase\u003c/p\u003e\n\u003cp\u003eCFU: Colony Forming Units\u003c/p\u003e\n\u003cp\u003eDAPG: 2,4 diacetylphloroglucinol\u003c/p\u003e\n\u003cp\u003eDTNB: 5,5′‑dithiobis‑(2‑nitrobenzoic acid)\u003c/p\u003e\n\u003cp\u003eDW: Dry Weight\u003c/p\u003e\n\u003cp\u003eEDTA: Ethylenediaminetetraacetic Acid\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eET: Ethylene\u003c/p\u003e\n\u003cp\u003eFW: Fresh Weight\u003c/p\u003e\n\u003cp\u003eGAE: Gallic Acid Equivalents\u003c/p\u003e\n\u003cp\u003eGFP: Green Fluorescent Protein\u003c/p\u003e\n\u003cp\u003eGSH: Reduced Glutathione\u003c/p\u003e\n\u003cp\u003eHCN: Hydrogen Cyanide\u003c/p\u003e\n\u003cp\u003eIAA: Indol Acetic Acid\u003c/p\u003e\n\u003cp\u003eISR: Induced Systemic Resistance\u003c/p\u003e\n\u003cp\u003eITS: Internal Transcribed Spacer\u003c/p\u003e\n\u003cp\u003eJA: Jasmonic Acid\u003c/p\u003e\n\u003cp\u003eMDA: Malondialdehyde\u003c/p\u003e\n\u003cp\u003eNA: Nutrient Agar\u003c/p\u003e\n\u003cp\u003eNB: Nutrient Broth\u003c/p\u003e\n\u003cp\u003eNBT: Nitrobluetetrazolium\u003c/p\u003e\n\u003cp\u003eOD: Optical Density\u003c/p\u003e\n\u003cp\u003ePAL: Phenylalanine Ammonia Lyase\u003c/p\u003e\n\u003cp\u003ePCR: Polymerase Chain reaction\u003c/p\u003e\n\u003cp\u003ePDA: Potato Dextrose Agar\u003c/p\u003e\n\u003cp\u003ePGPR: Plant Growth Promoting Rhizobacteria\u003c/p\u003e\n\u003cp\u003ePIRG: Percentage Inhibition of Radial Growth\u003c/p\u003e\n\u003cp\u003ePMSF: Phenylmethylsulfonyl fluoride\u003c/p\u003e\n\u003cp\u003ePO: Peroxidase\u003c/p\u003e\n\u003cp\u003ePPO: Polyphenol Oxidase\u003c/p\u003e\n\u003cp\u003ePVP: Polyvinylpyrrolidone\u003c/p\u003e\n\u003cp\u003eQE: Quercetin Equivalents\u003c/p\u003e\n\u003cp\u003eSA: Salycilic Acid\u003c/p\u003e\n\u003cp\u003eSAR: Systemic Acquired Resistance\u003c/p\u003e\n\u003cp\u003eSOD: Superoxide Dismutase\u003c/p\u003e\n\u003cp\u003eTBA: Thiobarbituric Acid\u003c/p\u003e\n\u003cp\u003eTCA: Trichloroacetic Acid\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupple\u003c/strong\u003e\u003cstrong\u003ementary information is available online\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors are grateful to Biotechnology center (University of Yaounde 1) for providing instrumentation facility for carrying out the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was carried out in collaboration among all authors. Author NMSA investigated the work, performed methodology, wrote and prepared the original draft and helped in software development of the manuscript. Author KMLB and MNJ did conceptualization, reviewed and edited the manuscript. Author TNS did data validation, performed methodology, reviewed and edited the manuscript. Author NNRA administered and supervised the work. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during this study are available from authors on request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBacker, R., Rokem, J. S., Ilangumaran, G., Lamont, J., Praslickova, D., Ricci, E., Subramanian, S., \u0026amp; Smith, D. L. (2018). Plant Growth-Promoting Rhizobacteria: Context, Mechanisms of Action, and Roadmap to Commercialization of Biostimulants for Sustainable Agriculture. \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e, 1473. https://doi.org/10.3389/fpls.2018.01473\u003c/li\u003e\n\u003cli\u003eBates, L. S., Waldren, R. P., \u0026amp; Teare, I. D. (1973). Rapid determination of free proline for water-stress studies. \u003cem\u003ePlant and Soil\u003c/em\u003e, \u003cem\u003e39\u003c/em\u003e(1), 205\u0026ndash;207. https://doi.org/10.1007/bf00018060\u003c/li\u003e\n\u003cli\u003eBeauchamp, C., \u0026amp; Fridovich, I. (1971). Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. \u003cem\u003eAnalytical Biochemistry\u003c/em\u003e, \u003cem\u003e44\u003c/em\u003e(1), 276\u0026ndash;287. https://doi.org/10.1016/0003-2697(71)90370-8\u003c/li\u003e\n\u003cli\u003eBloemberg, G. V., \u0026amp; Lugtenberg, B. J. J. (2001). 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Induction of Phenylalanine Deaminase by Light and its Relation to Chlorogenic Acid Synthesis in Potato Tuber Tissue. \u003cem\u003ePlant Physiology\u003c/em\u003e, \u003cem\u003e40\u003c/em\u003e(5), 779\u0026ndash;784. https://doi.org/10.1104/pp.40.5.779\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Potato, Late blight, Biocontrol, Plant defense, Phenolic compounds","lastPublishedDoi":"10.21203/rs.3.rs-9088149/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9088149/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe oomycete \u003cem\u003ePhytophthora infestans\u003c/em\u003e, causal agent of potato late blight, remains a major constraint to global potato production due to its rapid evolution and resistance to fungicides and host resistance genes. While single-strain biocontrol agents offer sustainable alternatives, their inconsistent field performance limits widespread adoption. Here, we demonstrate that a rationally designed consortium comprising \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e BaC21 and \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e DS17R provides synergistic protection against late blight through multilayered mechanisms. From 22 rhizosphere \u003cem\u003eBacillus\u003c/em\u003e isolates, BaC21 was selected for its superior antagonism (18.80 mm inhibition zone, 91.66% volatile-mediated suppression, 76.8% cell-free filtrate activity). Co-inoculation enhanced root colonization of both strains, with \u003cem\u003eBacillus\u003c/em\u003e populations increasing 1.19-fold and \u003cem\u003ePseudomonas\u003c/em\u003e 1.11-fold over single applications. Under greenhouse conditions, the BDR consortium reduced disease severity by 80.8%, significantly outperforming single strains. Field trials confirmed efficacy comparable to the fungicide Ridomil, with BDR-treated plots yielding 24.8 t.ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e a three-fold increase over untreated controls. Mechanistically, the consortium induced temporally coordinated phytohormone signaling: early jasmonate/ethylene peaks (JA: 95 ng.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW at 12 h; ethylene: 6.0 nL.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW/h at 12 h) followed by sustained salicylate accumulation (550 ng.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW at 48 h, r = -0.92 with AUDPC), circumventing SA-JA antagonism. This primed state activated phenylpropanoid metabolism (PAL 2.98 \u0026micro;mol h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e protein), lignin deposition (38.5 mg.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW), and antioxidant capacity (cysteine 265 nmol.g\u003csup\u003e\u0026minus;1\u003c/sup\u003eFW) without compromising photosynthesis (chlorophyll maintained at 2.35 mg.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW). Synergy factors reached 1.50 for disease reduction and 1.62 for yield increase. This work establishes a mechanistic framework for rationally designing microbial consortia that integrate ecological, hormonal, and biochemical complementarity for sustainable crop protection.\u003c/p\u003e","manuscriptTitle":"Rationally designed Bacillus-Pseudomonas consortium with synergistic control of potato late blight through biochemical defenses and physiological optimization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-26 17:22:54","doi":"10.21203/rs.3.rs-9088149/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"17add245-7847-4bc7-9eb8-e5b8b247ff7b","owner":[],"postedDate":"March 26th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Reject after review","date":"2026-05-07T13:04:59+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-07T17:05:28+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-26 17:22:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9088149","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9088149","identity":"rs-9088149","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}